Method of assessing risk of pml

ABSTRACT

The invention relates to methods of assessing a patient&#39;s risk of developing Progressive multifocal leukoencephalopathy (PML).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/491,810, filed May 31, 2011, U.S. Provisional Application No.61/508,584, filed Jul. 15, 2011, U.S. Provisional Application No.61/550,257, filed Oct. 21, 2011, and U.S. Provisional Application No.61/636,588, filed Apr. 20, 2012. The prior applications are incorporatedherein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods of assessing a patient's risk ofdeveloping Progressive multifocal leukoencephalopathy (PML).

BACKGROUND OF INVENTION

The anti-VLA-4 (Very Late Antigen 4) antibody therapeutic natalizumab isindicated to treat relapsing forms of multiple sclerosis (MS) andmoderate-to-severe Crohn's Disease. Natalizumab treatment, however, isassociated with an increased risk of progressive multifocalleukoencephalopathy (PML), an opportunistic brain infection caused bythe JC virus (JCV). PML occurs primarily in immunocompromisedindividuals and in patients receiving certain immunomodulatorytherapies, including natalizumab. PML is hypothesized to be the resultof a complex interaction between host and viral factors, leading toreactivation and mutation of latent archetype JCV to a neurotrophic formwhich can infect oligodendrocytes in the central nervous system.

SUMMARY OF INVENTION

The invention relates, inter alia, to an optimized analyticallyvalidated, sensitive assay for detecting the presence of JCV antibodiesin a biological fluid, e.g., serum or plasma and to various othermethods, including methods of evaluating and/or treating patients.

Accordingly, in one aspect, the invention features, a method ofevaluating the level of anti-JCV antibody in a sample. The methodcomprises one or more or all of the following steps:

(a) forming a first reaction mixture comprising a first aliquot ofsample and a substrate on which is disposed HPVLP (Highly PurifiedViral-Like Particle, e.g., Highly Purified VP1 Particle), e.g., a highsignal-to-noise HPVLP substrate;

(b) detecting the level of anti-JCV antibody bound to said substrate onwhich is disposed HPVLP, e.g., a high signal-to-noise HPVLP substrate,e.g., by detecting a labeled detection reagent, e.g., an enzyme labeledanti-IgG antibody, bound to anti-JCV antibody bound to said substrate;thereby evaluating the level of anti-JCV antibody in a sample (as isdiscussed herein, the method can comprise classifying, or assigning, tothe sample, a value indicative of the level of anti-JCV antibody, whichvalue is sometimes referred to herein as an index value. The value canbe used to evaluate the sample or a patient and in embodiments, todetermine whether to proceed to an additional step of the method, e.g.,step (c) below); and

(c) forming a second reaction mixture containing a second aliquot ofsample and solution-phase HPVLP, and detecting the level of unboundanti-JCV antibody in said second reaction mixture, such as by detectinganti-JCV antibody capable of binding with a substrate on which isdisposed HPVLP, e.g., a high signal-to-noise HPVLP substrate (as isdiscussed herein, the method can comprise classifying, or assigning, tothe sample, a value indicative of the degree to which incubation withthe soluble-phase HPVLP reduces the level of unbound anti-JCV antibodyin the second reaction mixture, which value is sometimes referred toherein as inhibition, % inhibition, or the like. This value can be usedto evaluate the sample or a patient),

thereby evaluating the level of anti-JCV antibody in a sample.

In an embodiment the method further comprises:

(d) forming a third reaction mixture containing a third aliquot underconditions where anti-JCV antibodies in the sample are not bound byHPVLP or other antigen, and detecting the level of anti-JCV antibody inthe third reaction mixture, such as by detecting anti-JCV antibodycapable of binding with a substrate on which is disposed HPVLP, e.g., ahigh signal-to-noise HPVLP substrate. The inhibition or % inhibition canbe calculated as a function of the degree that incubation withsoluble-phase HPVLP (step (c)) reduces the amount of unbound anti-JCVantibody, as compared to the result in step (d).

In an embodiment the method comprises steps (a) and (b), and optionally,providing the results to another entity, e.g., a healthcare provider.

In an embodiment the method comprises steps (a), (b), and (c), andoptionally, providing the results to another entity, e.g., a healthcareprovider.

In an embodiment the method comprises steps (a), (b), (c), and (d), andoptionally, providing the results to another entity, e.g., a healthcareprovider.

In an embodiment the method comprises step (c) and optionally providingthe results to another entity, e.g., a healthcare provider.

In an embodiment the method comprises step (c) and (d), and optionally,providing the results to another entity, e.g., a healthcare provider.

Methods described herein use optimized levels and amounts of reagents,allowing for improved performance. Thus, in an embodiment, for the firstreaction mixture, 20 ngs to 60 ngs, 30 ngs to 50 ngs, 20 ngs to 40 ngs,35 ngs to 45 ngs of HPVLP are disposed on said substrate. In anembodiment about 20 ngs, 30 ngs, 40 ngs, 50 ngs or 60 ngs of HPVLP aredisposed on said substrate. Typically, a multi-substrate device, e.g., amulti-well plate, e.g., a polystyrene multi-well plate, will have anamount of HPVLP specified herein on each of a plurality of substrates. Atypical substrate is the interior of a well on a multi-well plate.

Methods described herein use optimized ratios of reagents and sample,allowing for improved performance. In an embodiment the ratio of μl ofsample (this refers to undiluted sample, or the amount of sample in adilution, so 100 μl of a 1:100 μl dilution would be 1 μl of sample),e.g., serum or plasma, to ngs of HPVLP disposed on the substrate in thefirst reaction is: between 1:100 and 1:20; 1:80 and 1:30; 1:60 and 1:20;1:20 and 1:60; 1:30 and 1:50. In an embodiment the ratio of μl ofsample, e.g., serum or plasma, to ngs of HPVLP disposed on the substrateis about: 1:30, 1:40, or 1:50. In an embodiment the ratio of μl ofsample, e.g., serum or plasma, to ngs of HPVLP disposed on the substrateis about: (0.08 to 1.2): 30, (0.08 to 1.2): 40, or (0.08 to 1.2): 50.

In one embodiment, the sample, e.g., serum, for the first reaction isdiluted, such as by about 100-fold, in buffer, for example, prior tocontact with the substrate on which is disposed HPVLP, e.g., a highsignal-to-noise HPVLP substrate. In one embodiment, detection is with anenzyme labeled antibody, e.g., an enzyme labeled IgG, such as an HRP(Horseradish Peroxidase) labeled IgG. In another embodiment, thedetection reagent, e.g., an HRP labeled IgG, is added at a concentrationof at least 0.01 μg/mL, 0.02 μg/mL, 0.03 μg/mL, 0.04 μg/mL, 0.05 μg/mL,0.06 μg/mL, or 0.08 μg/ml. In one embodiment, the detection reagent isprovided at 10× to 100× excess over antibody bound to the substrate. Inan embodiment the detection reagent is provided, in an amount that givesequal to or more than 10×, 20×, 50×, 75× or 100×) excess as compared tothe antibody bound to the substrate.

In an embodiment the solution-phase HPVLP in (c) is present at 2× to100× excess particles over anti-JCV antibody in the second reactionmixture or sample. In an embodiment the excess of particles over theanti-JCV antibody in the second reaction mixture or sample is equal toor greater than 2×, 4×, 5×, 10×, 15×, 20×, 40×, 50×, 70×, 80×, 100× or110×.

In an embodiment, for the second reaction mixture, 20 ngs to 60 ngs, 30ngs to 50 ngs, 20 ngs to 40 ngs, 35 ngs to 45 ngs of HPVLP are disposedon said substrate. In an embodiment about 20 ngs, 30 ngs, 40 ngs, 50 ngsor 60 ngs of HPVLP are disposed on said substrate. Typically, amulti-substrate device, e.g., a multi-well plate, e.g., a polystyrenemulti-well plate, will have an amount of HPVLP specified herein on eachof a plurality of substrates. A typical substrate is the interior of awell on a multi-well plate.

In an embodiment, for the second reaction mixture, the sample iscontacted with the soluble-phase HPVLP and then unbound anti-JVCantibody is allowed to bind to a HPVLP disposed on a substrate. In anembodiment, for the second reaction mixture, the sample is insimultaneous contact with the soluble-phase HPVLP and HPVLP disposed ona substrate.

In an embodiment the ratio of μl sample (this refers to undilutedsample, or the amount of sample in a dilution, so 100 μl of a 1 μl:100μl dilution would be 1 μl of sample), e.g., serum or plasma, to ngs ofHPVLP disposed on the substrate is: between 1:100 and 1:20; 1:80 and1:30; 1:60 and 1:20; 1:20 and 1:60; 1:30 and 1:50. In an embodiment theratio of μl of sample, e.g., serum or plasma, to ngs of HPVLP disposedon the substrate is about: 1:30, 1:40, or 1:50. In an embodiment theratio of μl of sample, e.g., serum or plasma, to ngs of HPVLP disposedon the substrate is about: (0.08 to 1.2):30, (0.08 to 1.2):40, or (0.08to 1.2):50.

In one embodiment, the sample, e.g., serum, is diluted, such as by about100-fold, in, for example, buffer, prior to contact with the substrateon which is disposed HPVLP, e.g., a high signal-to-noise HPVLPsubstrate. In one embodiment, detection is with an enzyme labeledantibody, e.g., an enzyme labeled IgG, such as an HRP labeled IgG. Inanother embodiment, the detection reagent, e.g., an HRP labeled IgG, isadded at a concentration of at least 0.01 μg/mL, 0.02 μg/mL, 0.03 μg/mL,0.04 μg/mL, 0.05 μg/mL, 0.06 μg/mL, or 0.08 μg/ml. In one embodiment,the detection reagent is provided at 10× to 100× excess over antibodybound to the substrate. In an embodiment the detection reagent isprovided, in an amount that gives equal to or more than 10×, 20×, 50×,75× or 100×) excess as compared to the antibody bound to the substrate.

In one embodiment, responsive to the level of anti-JCV antibodiesdetected in step (b), steps (c) and/or (d) are performed.

In one embodiment, responsive to the level of anti-JCV antibodiesdetected in step (b), e.g., the index level (nOD) is >0.2 and is <0.4,then steps (c) and (d) are performed.

In one embodiment, the sample, e.g., serum or plasma, is diluted, suchas by an amount equal to or greater than about 50, 100, or 150 fold, in,e.g., buffer, prior to forming said second reaction mixture. In anotherembodiment, the sample, e.g., serum or plasma, is diluted, such as by anamount equal to or greater than about 50-fold, 100-fold, or 150-fold,in, e.g., buffer, prior to forming said third reaction mixture. Inanother embodiment, detection of one or both of the second and thirdreaction mixture is with an enzyme labeled antibody, e.g., an enzymelabeled IgG, e.g., an HRP labeled IgG.

Detection of one or both of the second and third reaction mixtures canbe with an HRP labeled IgG, added at a concentration of at least 0.01,0.02, 0.03, 0.04, 0.05, 0.06, or 0.08 μg/ml. In one embodiment, thedetection reagent is provided at 10× to 100× (e.g., 10×, 20×, 50×, 75×or 100×) excess as compared to the antibody bound to the substrate.

In one embodiment evaluating the level of anti-JCV antibody in a samplefurther includes evaluating a standard, such as a cut off calibrator,having, e.g., an index of about 1 (e.g., a optical density of 1, where apositive control has an optical density of 1.3, and a negative controlhas an optical density of 0.1) and a signal-to-noise ratio of equal toor greater than 15× to 20× (e.g., equal to or greater than 16×, 17×,18×, or 19×), in step b. Another embodiment includes evaluating astandard, e.g., a positive control, having, for example, a score ofabout 1.3, in step b. In other embodiments, the method further includesevaluating a standard, such as a negative control, having, e.g., a scoreof about 0.1, in step b.

In an embodiment the method includes determining the amount that bindingto said soluble phase HPVLP particles inhibits or reduces binding tosubstrate disposed HPVLP particles as compared with binding to substratedisposed HPVLP particles in said first aliquot. The results of the firststep of the two-step assay (steps (a) and (b) above), are typicallyexpressed as a normalized OD (nOD, or “index”) value. The results of thesecond step of the two-step assay (steps (c) and optionally (d) above),are typically expressed as “percent inhibition.” In an embodiment thenOD is OD₄₅₀. In an embodiment said inhibition is less than or equal toa predetermined value, e.g., 45%, and said sample is classified asnegative.

In an embodiment said inhibition is greater than a predetermined value,e.g., 45% and said sample is classified as positive.

In one embodiment, a cut-off calibrator (CO) is adjusted to have areactivity index (nOD) of about 1.0, and a positive control (PC) isadjusted to have a reactivity index of about 1.3. The CO and PCsolutions are made by mixing a serum positive for JCV antibodies with aserum that is negative for JCV antibodies. For the negative control(NC), which can be, for example, a bottle of anti-JCV antibody-negativesera, the index (nOD) target is about 0.1.

In one embodiment the JCV antigen is a VLP particle, such as a HPVLPthat is chromatographically purified prior to use in an assay featuredin the invention.

In certain embodiments, the sample is a serum sample, a urine sample, aplasma sample, a blood sample or a cerebrospinal fluid (CSF) sample. Inone embodiment the sample is a serum sample diluted 1:101 prior toforming the first reaction mixture comprising a first aliquot of thesample and the substrate on which is disposed HPVLPs.

In another embodiment, the secondary detection reagent (e.g., ananti-human IgG) is conjugated to a detectable agent, such as aperoxidase, such as HRP. In one embodiment, the secondary detectionreagent can be anti-human IgG, wherein the anti-human IgG is conjugatedto HRP. In another embodiment, the detection reagent solution containingIgG-HRP is used at 0.04 μg/mL. For example, a 0.8 mg/mL stock solutionof IgG-HRP is diluted 1:15,000, 1:20,000, 1:30000 or more, prior to usein the assay to detect the level of anti-JCV antibody bound to HPVLP. Inanother embodiment, the concentration of the secondary detection reagentis adjusted for new lots to match signal to previous lot and theincubation time with the conjugate is only 30 min. In one embodiment,TMB (tetramethylbenzidine) and hydrogen peroxide in buffer are incubatedwith the reaction mix containing the HRP IgG mixture bound to anti-JCVantibody for 20 minutes, ±2 minutes.

In an embodiment a decrease in the detected level in the secondary assaysample compared to the sample that was not preincubated indicates thesample is positive for anti-JCV antibody, and a change in the detectedlevel below a specified percentage indicates that there is noJCV-specific antibody present in the sample.

In one embodiment, the sample is determined to have an index value(i.e., nOD value) >0.2 and <0.4 (the “indeterminant zone”) after thefirst step of the assay, which is the formation of a first reactionmixture comprising a first aliquot of sample and a substrate on which isdisposed HPVLP, e.g., a high signal-to-noise HPVLP substrate, anddetecting the level of anti-JCV antibody bound to said substrate onwhich is disposed HPVLP, e.g., a high signal-to-noise HPVLP substrate. Asecond aliquot of the sample can then be tested in the second step ofthe assay, which comprises formation of a second mixture between thesecond aliquot and a solution-phase HPVLP prior to detecting unboundanti-JCV antibody in the second mixture by contacting the second mixturewith a substrate on which is disposed HPVLP, e.g., a highsignal-to-noise HPVLP substrate.

In another embodiment, if the sample is determined to have an indexvalue <0.2 after the first step of the assay, then the sample isdetermined to be anti-JCV antibody negative. In one embodiment, a sampledetermined to be anti-JCV antibody negative is not evaluated using thesecond step of the assay.

In another embodiment, if the sample is determined to have an indexvalue >0.4 after the first step of the assay, then the sample isdetermined to be anti-JCV antibody positive. In one embodiment, a sampledetermined to be anti-JCV antibody positive is not evaluated using thesecond step of the assay.

In one embodiment, the invention comprises obtaining a biological samplefrom a subject (e.g., plasma, serum, blood, urine, or cerebrospinalfluid (CSF)); and correlating the detected level with a reference, suchthat the reference is selected to indicate a false negative rate notgreater than 3% and minimal cross reactivity to other polyoma viruses,e.g., BK virus (BKV). In some embodiments, the reference, derived from acontrol sample or set of samples, is processed with the sample from thesubject. In some embodiments, the reference is selected such that thefalse negative rate of the assay is not greater than 1%. The assay canbe performed such that the HPVLP is disposed on a solid substrate suchas a microtiter plate or slide. In some embodiments, the HPVLP consistsessentially of VP1 viral protein. The HPVLP can further include otherviral proteins, for example at least one of a VP2, or a VP3. The viralprotein(s) in the HPVLP can be recombinantly derived (e.g., a MAD1 VP1)or can be a naturally-occurring viral protein (e.g., derived from anaturally-occurring source). The method can be performed using, forexample, a biological sample obtained from a subject currently beingtreated with an immunomodulatory drug, a subject considering initiatingtreatment with an immunomodulatory drug, or a subject suspected ofhaving Progressive Multifocal Leukoencephalopathy (PML).

In another aspect, the invention features, a kit containing a substrateon which is disposed HPVLP, e.g., a high signal-to-noise HPVLPsubstrate. The substrate can include a multiwall plate, such as a 96well plate. In one embodiment, the kit includes one or more or all ofthe following: a substrate, such as a plate with wells coated with JCVantigen substrate, e.g., HPVLP; a JCV antigen, e.g., HPVLP, lyophilizedor in solution; a JCV cut-off calibrator, an anti-JCV antibody positivecontrol and a JCV negative control, which are samples of sera, such ashuman sera. In one embodiment, the kit includes, or further includes oneof more reagents for detecting a complex containing anti-JCV antibodiesbound to antigen, and the reagents include, for example, a JCVconjugate, a casein sample, a detectable reagent, such as TMB(tetramethylbenzidine), a wash buffer, and a stop reagent.

In another aspect, the invention features, a substrate on which isdisposed HPVLP, e.g., a high signal-to-noise HPVLP substrate.

In another aspect, the invention features, a kit comprising an HPVLP andat least one reagent for performing an assay to identify an anti-JCVantibody level in a sample, such as a biological sample.

In other aspects, the invention relates to a solution comprising HPVLPparticles, consisting essentially of VP1-containing particles that aregreater in size than a VP1 pentamer (capsomere), e.g., containing about5, 10, 20, 30, 40, 50, 60, 70 or 72 pentamers or containing about 360VP1 molecules.

Another aspect of the invention is a method of preparing a solution ofHPVLPs, the method comprising removing VP1-containing particles from thesolution that are the size of a VP1 pentamer or less.

The methods disclosed herein are based at least in part on the discoverythat anti-JCV antibody titer and other characteristics such asaffinity/avidity can be indicators of a patient's risk of developingProgressive Multifocal Leukoencephalopathy (PML).

accordingly, in another aspect, the invention features, a method ofevaluating a patient's risk of developing PML, comprising acquiringknowledge of a JC Virus (JCV) antibody titer (e.g., determined asdescribed herein and expressed as normalized optical density (nOD) orindex) or affinity/avidity (e.g., as determined as described herein andexpressed percent inhibition in the confirmation step of the assay) in asample of the patient, and optionally comparing the value or valuesacquired with a reference disclosed herein, to thereby evaluate risk.

In one embodiment, an anti-JCV antibody titer or percent inhibition isdetermined in a biological sample from a patient, such as a blood (serumor plasma), or CSF sample.

If the titer or/and percent inhibition, or a function of both values isdetermined to be below a pre-determined level, the patient is determinedto be at a lower risk of developing PML, and if the titer and/or percentinhibition, or a function of both values is determined to be at or abovethe pre-determined level the patient is determined to be at a higherrisk of developing PML.

The method can further provide that determining the anti-JCV antibodytiter or percent inhibition in a sample of the patient requires removinga biological sample from the patient's body or analyzing a sample fromthe patient, or that if the patient is determined to be at a lower riskof developing PML, a therapy, such as immunosuppressant therapy isadministered to the patient.

In one embodiment, an anti-JCV antibody titer or percent inhibition isdetermined in more than one biological sample from a patient, such asone or more of a blood (serum or plasma), or CSF sample.

In one embodiment, the subject has multiple sclerosis, e.g., a multiplesclerosis patient that is already receiving therapy with an anti-VLA-4antibody, e.g., natalizumab. In one embodiment, the patient isdetermined to be at a lower risk of developing PML, and the patient isfurther administered an anti-VLA-4 therapy, such as an anti-VLA-4antibody, such as natalizumab, or a fragment thereof (such as anantigen-binding fragment thereof).

In one embodiment, the patient is determined to be at a higher risk ofdeveloping PML, and the patient is identified as someone who shouldreceive an alternative therapy, e.g., the patient should stop receivinganti-VLA-4 antibody therapy, e.g., natalizumab, and, e.g., receive analternative therapy, e.g., an alternative approved multiple sclerosis(MS) therapy such as Avonex®. In another embodiment, the patient isdetermined to be at a higher risk of developing PML, and the patient isadministered an anti-VLA-4 antibody therapy, e.g., natalizumab.

In one embodiment, the patient is determined to be at a higher risk ofdeveloping PML based upon anti-JCV antibody titer or percent inhibition,and the patient is identified as someone who should receive additionaltesting to determine risk of developing PML.

In one embodiment, the patient is determined to have a lower risk of PMLif, (i) the anti-JCV antibody titer as indicated by index value or nODis determined to be <0.5, or (ii) the anti-JCV antibody titer asindicated by index value or nOD is determined to be >0.5 and <3.0, andthe percent inhibition is determined to be less than or equal to 70% or60%. The patient is determined to have an intermediate risk of PML if,(i) the anti-JCV antibody titer as indicated by index value or nOD isdetermined to be >0.5 and <1.5, and the percent inhibition value isdetermined to be >70%. The patient is determined to have a higher riskof PML if, (i) the anti-JCV antibody titer as indicated by index valueor nOD is determined to be >0.5 and the percent inhibition value isdetermined to be >70%, or (ii) the patient showed an increase in index,nOD or titer by 2-fold from a previous test. The percent inhibition ofanti-JCV antibodies can be measured, for example, by: (i) contacting abiological sample from the subject with HPVLPs in a solution underconditions suitable for binding of an anti-JCV antibody in the sample toan HPVLP; (ii) separating the JCV antibodies bound to HPVLP from thesolution to create a secondary sample; (iii) contacting the secondarysample with HPVLP under the same conditions as (i); and (iv) detectingthe level of anti-JCV antibody binding to HPVLP in the secondary sample.

In one embodiment, the anti-JCV antibody titer is measured by, e.g., (i)contacting the biological sample with HPVLPs under conditions suitablefor binding of an anti-JCV antibody in the sample to an HPVLP; (ii)detecting the level of anti-JCV antibody binding in the sample toHPVLPs; and (iii) correlating the detected level with a reference set.The reference set can be selected to indicate a false negative rate notgreater than a predetermined amount, such as 3%. In another embodiment,anti-JCV antibody titer is measured by a commercial platform, such as aVIDAS® assay (bioMérieux), or another alternative platform, such as asolution-phase method or a lateral flow method.

In one embodiment, the assay indicates that the biological sample doesnot contain JCV antibodies, and the assay then further includes: (iv)contacting a portion of the biological sample from the subject withHPVLP in a solution prior to step (i) and where the HPVLP of step (i) isattached to a solid substrate, such as to provide a secondary sample;(v) contacting the secondary sample with HPVLP under the same conditionsas (i); (vi) detecting the level of anti-JCV antibody binding to HPVLPin the secondary sample; and (vii) comparing the detected level ofanti-JCV antibody in the secondary sample to the level of binding in thebiological sample when incubated with the solution without HPVLP. Adecrease in the detected level in the sample pre-incubated with HPVLPcompared to the solution-incubated sample indicates that the sample ispositive for an anti-JCV antibody, and no change in the detected levelindicates that anti-JCV antibody is not present above background levelsin the sample.

In one embodiment, the assay indicates that the biological samplecontains JCV antibodies, and the patient is determined to be at higherrisk for PML.

In yet another embodiment, the patient is determined to have a lowerrisk of PML if, (i) the anti-JCV antibody titer as indicated by indexvalue or nOD is determined to be <0.5, or (ii) the anti-JCV antibodytiter as indicated by index value or nOD is determined to be >0.5 and<3.0, and the percent inhibition is determined to be less than or equalto 70%. The patient is determined to have a higher risk of PML if, (i)the anti-JCV antibody titer as indicated by index value or nOD isdetermined to be >3 and the percent inhibition value is determined tobe >70%, or (ii) the patient showed an increase in index, nOD or titerby 2-fold from a previous test.

In one embodiment, only index value (nOD) or only percent inhibition isused to determine risk of PML. For example, in one embodiment, thepatient is determined to have a lower risk of PML if the anti-JCVantibody titer as indicated by index value or nOD is determined to be<0.5, the patient is determined to have a higher risk if the anti-JCVantibody titer as indicated by index value or nOD is determined tobe >0.5 and <1.5, or the patient is determined to have an even higherrisk if the anti-JCV antibody titer as indicated by index value or nODis determined to be >1.5.

In one embodiment, the assay indicates that the biological sample doesnot contain JCV antibodies above a background level, and the patient isdetermined to be at lower risk for PML.

In another aspect, the invention features, a method for evaluating ortesting an assay procedure. An anti-JCV antibody assay can bereevaluated for effectiveness at a predetermined interval, such as every6 months or every year. In one exemplary proficiency assay, a collectionof samples, e.g., 30, 40 or 50 serum samples and 30, 40, or 50 plasmasamples are provided such as for evaluation by the current optimizedmethod and a preceding earlier-generation method. The concordancebetween the results is assessed and if the concordance is found to begreater than, e.g., 90% or 95%, the performance of the assay can bedetermined to be acceptable. In one embodiment, a panel of samples,e.g., containing 90, 100, 150 or more samples, with known anti-JCVantibody status is utilized to assess consistency of assay performanceover time. The concordance between the results is assessed and if theconcordance is found to be greater than, for example, 90% or 95%, theperformance of the assay can be determined to be acceptable. The panelof samples is patient sera available in sufficient volume to create asample bank.

In one aspect, an entity, e.g., a healthcare provider, acquiresinformation resulting from an anti-JCV antibody assay described herein,and responsive to the information, administers a treatment describedherein to the patient, e.g., a MS patient.

In another aspect, a JCV assay described herein is performed on apatient, and then the patient is treated, e.g., the MS patient istreated, based on the results of the assay.

The anti-JCV antibody titer or percent inhibition in a patient can bereevaluated at regular intervals, such as every 3 months, every 6months, or every 12 months or at longer intervals or more frequently. Anobserved increase in antibody titer or percent inhibition can indicatean increase in the patient's risk of developing PML. For example, anincrease of 2 fold or 3 fold in antibody titer (nOD or index) canindicate an increased risk of PML. A patient receiving an anti-VLA-4therapy, such as a natalizumab, may stop therapy with the anti-VLA-4therapy, and optionally begin therapy with an alternative agent, e.g.,an immunosuppressant other than an anti-VLA-4 therapy, or other thannatalizumab. An increase in titer may present differently in patientshaving a high baseline titer (e.g., at a more narrow range in range oftiter) than in patients having a low baseline titer.

In one embodiment, a patient receiving an anti-VLA-4 antibody, e.g.,natalizumab, can be monitored, e.g., every five, six, seven, eight,nine, ten, eleven, twelve, fifteen, twenty, thirty, forty months, foranti-JCV antibody titer and/or percent inhibition. In one embodiment, apatient is not re-evaluated for the presence of JCV antibodies, or foranti-JCV antibody titer or percent inhibition within one or two or threeweeks after having received plasmapheresis. In another embodiment, apatient is not re-evaluated for the presence of JCV antibodies, or foranti-JCV antibody titer or percent inhibition within one or two or threeweeks after having received intravenous immunoglobulin (IVIG) treatment.

The measure of anti-JCV antibody titer can be in terms of nOD or anindex value.

Evaluation of a patient as described herein can be conducted prior toadministration of an anti-VLA-4 therapy, or after the patient has begunan anti-VLA-4 therapy.

In one embodiment, a patient is determined to be at a lower risk of PML,such as by an assay described herein, and the patient is administered ananti-VLA-4 therapy. In another embodiment, the patient is determined tobe at a higher risk of PML and the patient is administered an anti-VLA-4therapy, e.g., an anti-VLA-4 antibody, such as natalizumab. In yetanother embodiment, the patient is determined to be at a higher risk ofPML and the patient is administered a therapy other than an anti-VLA-4therapy, such as an interferon, glatiramer acetate or a corticosteroid.

In one embodiment, the patient is determined to have an increased riskfor PML, and the patient accordingly stops receiving an anti-VLA-4therapy.

The patient can be monitored at regular intervals, e.g., every 3 months,every 6 months, every year, or more or less frequently, for a decreasein anti-JCV antibody titer or a decrease in percent inhibition of JCVantibodies. A decrease in anti-JCV antibody titer or a decrease inpercent inhibition of JCV antibodies can indicate that the patient has alowered risk of developing PML.

In one embodiment, the anti-JCV antibody titer or percent inhibition ofJCV antibodies is determined to be decreased below a pre-determinedlevel, even after having been elevated, then the patient can beadministered, or determined to be a candidate to receive treatment with,an anti-VLA-4 therapy. If the patient previously received an anti-VLA-4,then the patient's anti-VLA-4 therapy can reinstated. After reinstatingthe anti-VLA-4 therapy, the patient can be evaluated every 6 months orevery year for a decrease in antibody titer or a decrease in percentinhibition of JCV antibodies.

In one embodiment, after a patient is determined to be at a higher riskof PML, e.g., the patient is determined to have an anti-JCV antibodytiter as measured by nOD of >0.5, e.g., >1.0 or >1.5, then the patientis not tested for JCV status again. For example, the patient can stoptherapy with an anti-VLA-4 therapy such as natalizumab, and not betested again for anti-JCV antibody status.

In one embodiment, a method of evaluating a patient as described herein,such as to determine an anti-JCV antibody titer or percent inhibition,can further include assessing other measures of risk predictors. Forexample, a method of evaluating a patient can further include: (a)determining if the patient has received extended treatment with ananti-VLA-4 therapy (e.g., longer than 24 months); or (b) determining ifthe patient has received a specified non-anti-VLA-4 immunosuppressanttherapy (e.g., mitoxantrone or other therapies in the last 2, 3, 5 yearsor ever in the patient's life). The relative risk of PML for a patientwho has an anti-JCV antibody titer or percent inhibition above apre-determined level but has no specified prior immunosuppressant useand has not had an extended treatment with an anti-VLA-4 therapy is lessthan the relative risk of a patient who has an anti-JCV antibody titeror percent inhibition below a pre-determined level and has specifiedprior immunosuppressant use or an extended treatment with an anti-VLA-4therapy, which is less than the relative risk of a patient who has ananti-JCV antibody titer or percent inhibition above a pre-determinedlevel and has specified prior immunosuppressant use and extendedtreatment with an anti-VLA-4 therapy.

In one embodiment, the patient previously received an anti-VLA-4therapy, e.g., natalizumab, and in another embodiment, the patient isadministered an anti-VLA-4 therapy, based on an evaluation, e.g., anevaluation of anti-JCV antibody titer or percent inhibition. Forexample, as a result of the evaluation, the patient can be classified asa candidate for anti-VLA-4 therapy. In one embodiment, a patientclassified as a candidate for anti-VLA-4 therapy is further administeredthe therapy.

In some embodiment, factors to be included in the stratification modelare the patient's age or gender.

Method described herein can incorporate one or more factors into theevaluation of the patient. Accordingly, in another aspect, the inventionfeatures, a method of evaluating a patient, e.g., as a candidate toreceive treatment with an anti-VLA-4 therapy.

The method includes, for example, acquiring or determining a JC Virus(JCV) antibody titer and percent inhibition in a biological sample fromthe patient, e.g., by a method described herein. If the antibody titeror percent inhibition is determined to be below a pre-determined level,then the patient can be classified as being suitable for treatment witha first category of therapy, such as an anti-VLA-4 therapy, e.g.,natalizumab. If the antibody titer or percent inhibition is determinedto be at or above the pre-determined level the patient is classified asbeing suitable for a second category of therapy, e.g., interferon,glatiramer acetate or a corticosteroid. Acquiring an anti-JCV antibodytiter and percent inhibition in a sample of a patient may includeremoving a biological sample from the patient's body or analyzing asample from the patient. The method of evaluation may also includeadministering a therapy, such as from the first category (e.g.,natalizumab) or the second category (e.g., interferon, glatirameracetate or a corticosteroid), to the patient.

In yet another embodiment, the patient is determined to have a lowerrisk of PML if, (i) the anti-JCV antibody titer as indicated by indexvalue or nOD is determined to be <0.5, or (ii) the anti-JCV antibodytiter as indicated by index value or nOD is determined to be >0.5 and<3.0, and the percent inhibition is determined to be less than or equalto 70%. The patient is determined to have a higher risk of PML if, (i)the anti-JCV antibody titer as indicated by index value or nOD isdetermined to be >1.5 and the percent inhibition value is determined tobe >70%, or (ii) the patient showed an increase in index, nOD or titerby 2-fold from a previous test. The patient is determined to have anintermediate risk of PML if the anti-JCV antibody titer as indicated byindex value or nOD is determined to be >0.5 and <1.5, and the percentinhibition value is determined to be >70%.

As discussed above, methods of evaluating a patient can incorporate morethan one consideration or factor. Thus, methods of evaluating a patientcan further include:

(aa) determining if the patient has received extended treatment with ananti-VLA-4 therapy (e.g., longer than 24 months) and in embodimentsproviding a prior anti-VLA-4 therapy exposure classification; or

(bb) determining if the patient has received a specified non-anti-VLA-4immunosuppressant therapy (e.g., in the last 2, 3, 5 years or ever inthe patient's life), and in embodiments providing a priorimmunosuppressive exposure classification.

Typically, a patient who has an anti-JCV antibody titer or percentinhibition above a pre-determined level but has no specified priorimmunosuppressant use and has not had an extended treatment with ananti-VLA-4 therapy is classified as having less risk of developing PMLthan the relative risk of a patient who has an anti-JCV antibody titeror percent inhibition below a pre-determined level and has specifiedprior immunosuppressant use or an extended treatment with an anti-VLA-4therapy, which is less than the relative risk of a patient who has ananti-JCV antibody titer or a percent inhibition above a pre-determinedlevel and has specified prior immunosuppressant use and extendedtreatment with an anti-VLA-4 therapy.

In one embodiment, the patient has previously received an anti-VLA-4therapy. In another embodiment, the method includes administering ananti-VLA-4 therapy, e.g., natalizumab to the patient.

In one embodiment, the patient is classified as a candidate foranti-VLA-4 therapy, and the patient is further administered theanti-VLA-4 therapy.

Patients who have received an anti-VLA-4 therapy, such as natalizumabfor 24 months or less, who have not previously received animmunosuppressant therapy (other than anti-VLA-4 therapy), and who testnegative for exposure to JCV (e.g., negative for JCV antibodies)typically have the lowest risk for developing PML. Conversely, patientswho received anti-VLA-4 therapy for longer than 24 months, who havepreviously received an immunosuppressant therapy (other than ananti-VLA-4 therapy), and who test positive for exposure to JCV (e.g.,positive for JCV antibodies) typically have the highest risk fordeveloping PML.

A patient's risk level for PML can be assessed by evaluating one, or anytwo or all three of the identified risk factors. For example, a patient,e.g., a patient with multiple sclerosis (MS) who tests negative foranti-JCV antibody titer can be determined to be at a lower risk for PML.A patient at a lower risk for PML can have a risk of less than about0.2/1000 patients, e.g., ≤0.11/1000.

In an embodiment, a patient, e.g., a patient with MS, who has receivedan anti-VLA-4 therapy, such as natalizumab, for 24 months or less (e.g.,for 23 months, 22 months, 20 months, 15 months, 12 months, 6 months, 1month or less), and who has not previously received an immunosuppressanttherapy can be determined to be at a lower risk for PML. For example,the patient can be determined to have a risk of PML of about 0.54/1000patients. The patient can accordingly be determined to be a candidate toreceive further treatment with an anti-VLA-4 therapy, such asnatalizumab.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for longer than 24 months, such as for about 25 to 48months or more (e.g., 26, 28, 30, 36, 40, or 48 months or more), and whohas not previously received an immunosuppressant therapy can bedetermined to be at, or classified as having, a higher risk for PML. Apatient at a higher risk of PML can have a risk of ≥about 3.7/1000patients, e.g., about 1.37/1000 patients. The patient can accordingly bedetermined to be a candidate to receive further treatment with ananti-VLA-4 therapy, such as natalizumab.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for 24 months or less (e.g., for 24 months, 22 months,20 months, 15 months, 12 months, 6 months, 1 month or less), and who isdetermined to be negative for anti-JCV antibodies, or JCV nucleic acid,can be determined to be at, or classified as having, a lower risk forPML. For example, the patient can be determined to be at a risk of≤0.2/1000 patients. The patient can accordingly be determined to be acandidate to receive further treatment with an anti-VLA-4 therapy, suchas natalizumab.

In an embodiment, a patient who has not received prior treatment with animmunosuppressant (other than an anti-VLA-4 therapy), and who isdetermined to be negative for JCV, is determined to be at a lower riskfor PML, e.g., ≤0.2/1000 patients. The patient can accordingly bedetermined to be a candidate to receive further treatment with ananti-VLA-4 therapy, such as natalizumab.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for longer than 24 months (e.g., for 25 months, 26months, 28 months, 30 months, 35 months, 38 months, 40 months, 48 monthsor longer), and who has previously received an immunosuppressant therapyother than an anti-VLA-4 therapy can be determined to be at a higherrisk for PML. A patient at a higher risk for PML can have a risk ofabout 0.37/1000 or greater, e.g., about 4.3/1000 patients. The patientcan accordingly be determined not to be a candidate to receive furthertreatment with an anti-VLA-4 therapy, such as natalizumab, or can bedetermined to be a candidate to receive treatment with an anti-VLA-4therapy accompanied by more frequent monitoring. For example, a patientat higher risk for PML who receives therapy with an anti-VLA-4 therapycan receive more frequent monitoring for development of PML, then apatient at lower risk of PML.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for 24 months or less (e.g., for 24 months, 22 months,20 months, 15 months, 12 months, 6 months, 1 month or less), and who haspreviously received an immunosuppressant therapy other than ananti-VLA-4 therapy can be determined to be at a higher risk for PML. Forexample, the patient can be determined to have a risk of PML of0.66/1000 patients. The patient can accordingly be determined not to bea candidate to receive further treatment with an anti-VLA-4 therapy,such as natalizumab, or can be determined to be a candidate to receivetreatment with an anti-VLA-4 therapy accompanied by more frequentmonitoring. For example, a patient at higher risk for PML who receivestherapy with an anti-VLA-4 therapy can receive more frequent monitoringfor development of PML, then a patient at lower risk of PML.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for longer than 24 months (e.g., for 25 to 48 months,such as 26, 30, 36, 42 or 48 months or longer), and who is determined tobe positive for JCV, is determined to be at a higher risk for PML. Thepatient can accordingly be determined not to be a candidate to receivefurther treatment with an anti-VLA-4 therapy, or can be determined to bea candidate to receive treatment with an anti-VLA-4 therapy accompaniedby more frequent monitoring.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for longer than 24 months (e.g., for 25 to 48 months,such as 26, 30, 36, 42 or 48 months or longer), and who has not receivedprior treatment with an immunosuppressant (other than an anti-VLA-4therapy), and who is determined to be positive for JCV, and isdetermined to be at a higher risk for PML. For example, the patient canbe determined to have a risk of PML of 4/1000 patients. The patient canaccordingly be determined not to be a candidate to receive furthertreatment with an anti-VLA-4 therapy, or can be determined to be acandidate to receive treatment with an anti-VLA-4 therapy accompanied bymore frequent monitoring.

In an embodiment, a patient, e.g., an MS patient, who has received priortreatment with an immunosuppressant other than an anti-VLA-4 therapy,and who is determined to be positive for anti-JCV antibodies, or JCVnucleic acid, can be determined to be at a higher risk for PML. Thepatient can accordingly be determined not to be a candidate to receivedfurther treatment with an anti-VLA-4 therapy, or can be determined to bea candidate to receive treatment with an anti-VLA-4 therapy accompaniedby more frequent monitoring.

In an embodiment, a patient who has received prior treatment with animmunosuppressant other than an anti-VLA-4 therapy, and who isdetermined to be positive for anti-JCV antibodies, or JCV nucleic acid,and who has received an anti-VLA-4 therapy, such as natalizumab, forlonger than 24 months (e.g., for 25 to 48 months, such as 26, 30, 36, 42or 48 months or longer) can be determined to be at a higher risk forPML. For example, the patient can be determined to have a risk of9.8/1000 patients. The patient can accordingly be determined not to be acandidate to received further treatment with an anti-VLA-4 therapy, orcan be determined to be a candidate to receive treatment with ananti-VLA-4 therapy accompanied by more frequent monitoring.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for 24 months or less (e.g., for 24 months, 22 months,20 months, 15 months, 12 months, 6 months, 1 month or less), and who hasreceived prior treatment with an immunosuppressant other than ananti-VLA-4 therapy, and who is determined to be positive for JCV, can bedetermined to be at a higher risk for PML. For example, the patient canbe determined to have a risk of PML of 4.5/1000 patients. The patientcan accordingly be determined not to be a candidate to receive furthertreatment with an anti-VLA-4 therapy, or can be determined to be acandidate to receive treatment with an anti-VLA-4 therapy accompanied bymore frequent monitoring.

In an embodiment, a patient who has received an anti-VLA-4 therapy, suchas natalizumab, for 24 months or less (e.g., for 24 months, 22 months,20 months, 15 months, 12 months, 6 months, 1 month or less), and who hasnot received prior treatment with an immunosuppressant (other than ananti-VLA-4 therapy), and who is determined to be positive for JCV, canbe determined to be at a higher risk for PML. For example, the patientcan be determined to have a risk of PML of 0.35/1000 patients. Thepatient can accordingly be determined not to be a candidate to receivefurther treatment with an anti-VLA-4 therapy, or can be determined to bea candidate to receive treatment with an anti-VLA-4 therapy accompaniedby more frequent monitoring.

A patient determined to have a lower risk of developing PML can bedetermined to have a risk of ≤about 0.54/1000 patients, e.g.,≤0.25/1000, ≤0.2/1000, 0.19/1000, ≤0.15/1000, ≤0.11/1000, ≤0.1/1000,e.g., 0.3/1000, 0.25/1000, 0.2/1000, 0.19/1000, 0.15/1000, 0.11/1000, or0.1/1000 or lower. A patient determined to have a higher risk of PML canbe determined to have a risk of about 0.54/1000 or greater, e.g., about0.55/1000, about 0.60/1000, about 0.66/1000, about 1.2/1000, about1.37/1000, about 2.0/1000, about 2.5/1000, about 3.0/1000, about4.3/1000, about 5.0/1000, about 7.8/1000, about 8.0/1000, or higher. Forexample, a patient determined to have a higher risk of PML can bedetermined to have a risk of 0.3/1000, 0.35/1000, 0.5/1000, 0.66/1000,1.2/1000, 1.37/1000, 2.0/1000, 2.5/1000, 3.0/1000, 4.3/1000, 5.0/1000,7.8/1000, 8.0/1000 or higher.

In one embodiment, a patient who has received prior treatment with ananti-VLA-4 therapy for longer than 24 months, and who has not receivedprior therapy with an immunosuppressant other than anti-VLA-4 therapy,and who is determined to be JCV negative, is determined to be at lowerrisk of developing PML, and therefore a suitable candidate to receivefurther treatment with an anti-VLA-4 therapy, such as natalizumab.However, due to having received anti-VLA-4 therapy for longer than 24months, the risk assessment can include a recommendation to monitor thepatient more frequently for the development of adverse symptoms, such assymptoms that may indicate the development of PML.

Enhanced monitoring of patients for the development of PML can includeincreased frequency of tests to identify the presence of JCV, e.g.,increased testing by anti-JCV antibody assays or nucleic acid-basedassays. Enhanced monitoring can also include MRI scans to identify brainlesions due to PML.

In one embodiment, a patient who has anti-JCV antibodies at less than apreselected criterion has an undetectable level of anti-JCV antibodies.

In one embodiment, the patient has previously received an anti-VLA-4therapy, and in another embodiment, the patient has not previouslyreceived an anti-VLA-4 therapy.

In yet another embodiment, the patient is classified as a candidate foranti-VLA-4 therapy, and an anti-VLA-4 therapy, e.g., natalizumab, isadministered to the patient.

In one embodiment, making a determination, e.g., determining if thepatient is negative for JCV, requires providing (e.g., obtaining orreceiving) a biological sample from the patient, and performing animmunoassay, such as an ELISA assay to detect JCV antibodies in thesample. In another embodiment, a determination, e.g., determining if thepatient is negative for JCV requires providing a biological sample fromthe patient and performing an assay, such as a PCR-based assay, todetect JCV nucleic acid in the sample.

If the patient is classified as a candidate for anti-VLA-4 therapy, thepatient can be further administered an anti-VLA-4 therapy. A patientclassified as a candidate for anti-VLA-4 therapy is determined to have alower risk for developing PML, e.g., a risk of less than about 0.2/1000patients, e.g., 0.3/1000 patients, or 0.2/1000 patients or 0.19/1000patients or 0.11/1000 patients. For example, a patient having a lowerrisk of PML can have a risk of ≤0.2/1000.

A patient not classified as a candidate for anti-VLA-4 therapy, ordetermined to be a candidate for anti-VLA-4 therapy with enhancedmonitoring for development of PML, is determined to have a higher riskfor developing PML, e.g., a risk of greater than or equal to about0.3.7/1000 patients. For example, a patient determined to have a higherrisk of PML can have a risk of 0.37/1000, 0.35/1000, 0.66/1000,1.2/1000, 1.37/1000, 2.5/1000, 4.3/1000, or 7.8/1000 patients.

In an embodiment, a prior immunosuppressant exposure classification, ifselected, is one of the following:

a positive prior immunosuppressant exposure classification thatcorresponds to having received a non-anti-VLA-4 immunosuppressanttherapy within a preselected time period, e.g., within 1, 3, or 5 years,or in the patient's lifetime; and

a negative prior immunosuppressant exposure classification thatcorresponds to being free of a non-anti-VLA-4 immunosuppressant therapyfor a preselected time period, e.g., within 1, 3, or 5 years, or in thepatient's lifetime.

In an embodiment, a prior VLA-4 therapy exposure classification, ifselected, is one of the following:

a positive prior VLA-4 therapy exposure classification that correspondsto having received an anti-VLA-4 therapy for more than a preselectedperiod of time, e.g., as much or more than 1, 2, 3, or 5 years; and

a negative prior VLA-4 therapy exposure classification that correspondsto having received an anti-VLA-4 therapy for less than a preselectedperiod of time, e.g., less than 6 months, 1, 2, 3, or 5 years

In an embodiment, the method comprises providing a treatment suitabilityclassification, which, e.g., can be selected from one of:

a positive treatment suitability classification that is correlated withsuitability of the patient for anti-VLA-4 treatment (the positivetreatment suitability classification can be further subdivided intopositive treatment suitability classifications that are accompanied byvarious warnings or requirements for monitoring, such as increasedmonitoring for development of PML); and

a negative treatment suitability classification that is correlated withunsuitability of the patient for anti-VLA-4 treatment, or suitability ofthe patient for anti-VLA-4 treatment, accompanied by various warnings orrequirements for increased monitoring, such for development of PML.

A positive treatment suitability classification correlates with a lowerrisk of developing PML, and a negative treatment suitabilityclassification correlates with a higher risk of developing PML. A lowerrisk of developing PML typically corresponds to a risk less than0.2/1000 patients, and a higher risk of developing PML corresponds to arisk of ≥0.37/1000.

If the patient is assigned a low exposure classification, and a negativeJCV status classification, the patient is assigned a positive treatmentsuitability classification, e.g., a modified positive treatmentsuitability classification that advises or requires monitoring fordevelopment of PML.

If the patient is assigned a negative prior immunosuppressant exposureclassification, and a negative anti-JCV antibody status classification,the patient can be assigned a positive treatment suitabilityclassification, e.g., a modified positive treatment suitabilityclassification that advises or requires monitoring for development ofPML.

If the patient is assigned a low exposure classification, a negativeprior immunosuppressant exposure classification, and a negative JCVantibody classification, the patient is assigned a positive treatmentsuitability classification.

In one embodiment, the patient is assigned a positive treatmentsuitability classification, and the patient is further administered ananti-VLA-4 therapy, e.g., natalizumab.

In one aspect, a method of evaluating a patient, e.g., evaluating apatient's risk of developing PML, is also provided. The method includes:

(aaa) determining if the patient is negative or positive for JCV, suchas by determining whether the level of anti-JCV antibodies is less thanor greater than a preselected criterion, e.g., as determined by a methoddisclosed herein;

(bbb) determining if the patient has received an anti-VLA-4 therapy forgreater than a preselected period of time (e.g., longer than 24 months),or less than a preselected period of time, e.g., 24 months or less, orhas not received anti-VLA-4 therapy in a preselected period, e.g., inthe last 2, 3, 5 years, or ever in the patient's life;

(ccc) determining if the patient has been free of a non-anti-VLA-4immunosuppressant therapy for a preselected period of time or hasreceived a non-anti-VLA-4 immunosuppressant therapy for a preselectedperiod of time (a specified time) (e.g., the last 1, 2, 3, 4, 5, or 10years, or ever in the patient's life); and responsive to thedeterminations, evaluating the patient.

In an embodiment, responsive to a determination that the patient isnegative for JCV, determining that a patient is at a lower risk ofdeveloping PML.

In an embodiment, responsive to a determination that the patient ispositive for JCV, determining that the patient has a higher risk of PML.

In an embodiment a determination, e.g., determining that the patient isnegative for JCV, comprises or requires removing a sample from thepatient's body or analyzing a sample from the patient, or the methodfurther requires administering a therapy to the patient. The therapycan, e.g., in the case of a lower risk patient, be an anti-VLA-4 therapy(e.g., anti-VLA-4 antibody), or, e.g., in the case of a lower riskpatient, an alternative (non-anti-VLA-4) therapy, e.g., an interferon,glatiramer acetate or a corticosteroid.

In one aspect, a method of complying with instructions is provided. Theinstructions may, for example, appear on a government required packageinsert, e.g., an FDA (Food and Drug Administration) or EMA (EuropeanMedicines Agency) mandated package, and provide guidance for the use ofan anti-VLA-4 therapy. The method of complying with instructionsincludes, optionally receiving the instructions; acquiring the resultsof an evaluative method described herein, and responsive to the acquiredresult, providing a recommendation for therapy to a patient, andoptionally, further administering a therapy to the patient. Theinstruction can specify an evaluative method as described herein isessential for safely administering the therapy. The therapy may be ananti-VLA-4 therapy, e.g., natalizumab.

A method of evaluating a patient is provided, where the method requiresproviding a kit for the collection or transport of a patient sample to ahealthcare provider; receiving a patient sample from the healthcareprovider; performing a method as claimed herein.

A method of treating a patient is also provided. The method requiresacquiring the result of a patient or sample evaluation method describedherein, and responsive to the acquired result, administering a therapyto the patient. The therapy can be an anti-VLA-4 therapy, such asnatalizumab. E.g., For example, responsive to the results of steps (a)and (b), steps (a), (b), and (c), steps (a), (b), (c) and (d), step (c),or steps (c) and (d) administering a therapy, e.g., a therapy describedherein, to the patient.

A computerized method of authorizing reimbursement, such as for the costof an anti-VLA-4 therapy, is also provided. The party to be reimbursedmay be a third party payor, such as an insurance company or governmentalagency. The method can include (a) acquiring the result of a patientevaluation method described herein, and recording the result on acomputer readable medium; (b) acquiring evidence of administration of ananti-VLA-4 therapy to the patient and recording the evidence on acomputer readable medium; and (c) if the result is consistent withadministration of the anti-VLA-4 therapy, authorizing reimbursement to,or reimbursing, the party.

In one aspect, a method is provided for selecting or classifying apatient as a candidate to receive treatment with an anti-VLA-4 therapy,e.g., natalizumab. For example, the method can include determining thata patient has previously received an anti-VLA-4 therapy for 24 months orless, e.g., for 1 to 24 months, 2 to 20 months, 5 to 15 months, or 10 to12 months, or that a patient has not previously received treatment withan immunosuppressant, and assessing anti-JCV antibody titers or percentinhibition. In one embodiment, assessing involves analyzing a samplefrom the patient. The sample can be, for example, a sample of blood,plasma, serum, urine, or cerebrospinal fluid. If the assessmentindicates that the patient is positive for JCV, e.g., positive foranti-JCV antibodies or JCV nucleic acid, then the patient is notselected or classified as a candidate for treatment with the anti-VLA-4therapy. If the assessment indicates that the patient is negative forJCV, e.g., negative for anti-JCV antibodies or JCV nucleic acid, thenthe patient is selecting or classified as a candidate to receivetreatment with the anti-VLA-4 therapy.

An assay for the presence of anti-JCV antibodies can be an immunoassay,such as an ELISA assay. An assay for JCV nucleic acid can be, e.g., aPCR assay or a Next Generation Sequencing (NGS) method.

A patient determined to be at lower risk for PML can further beadministered an anti-VLA-4 therapy, such as natalizumab. A patientdetermined to be at higher risk for PML can further be administered analternative to an anti-VLA-4 therapy, such as an interferon, glatirameracetate, a corticosteroid or a TNF agonist. In one embodiment, a patientdetermined to be at higher risk for PML can be further administered ananti-VLA-4 therapy, and can be required to receive an increasedfrequency of testing for PML, and where the patient is initiallydetermined to be JCV negative, can also be required to received anincreased frequency of testing for JCV.

In one aspect, a method of determining a patient's risk for PML isprovided. The method includes (a) determining that a patient haspreviously received an anti-VLA-4 therapy (e.g., natalizumab) for 24months or less, or that a patient has not previously received treatmentwith an immunosuppressant; and (b) assessing a patient's anti-JCVantibody status, where the assessing step includes analyzing a samplefrom the patient. If the assessment indicates that the patient is JCVnegative, then the patient is determined to be at lower risk for PML. Ifthe assessment indicates that the patient is JCV positive, then thepatient is determined to be at higher risk for PML.

In another aspect, a method of treating a patient is provided. Thetreatment method includes, e.g., determining the patient's priorexposure to an anti-VLA-4 therapy, and determining whether the patientpreviously received treatment with an immunosuppressant. Optionally, thepatient's status for JCV can also be determined.

If the patient is determined to have received the anti-VLA-4 therapy for24 months or less, and not to have previously received treatment with animmunosuppressant, then the patient is determined to be at lower riskfor PML, and the patient is administered the anti-VLA-4 therapy. If thepatient is determined to have received natalizumab for longer than 24months (e.g., 25 months or longer), and not to have previously receivedtreatment with an immunosuppressant, then the patient is determined tobe at higher risk for PML, and the patient is administered analternative to anti-VLA-4 therapy, e.g., an interferon, acorticosteroid, a statin or a TNF antagonist.

Determining the patient's prior exposure to an anti-VLA-4 therapy or animmunosuppressant can include asking the patient or a caregiver, e.g., aphysician, nurse, parent or other caregiver. In some cases, determiningthe patient's prior exposure can include accessing the information in adatabase, e.g., a database of medical records.

Also provided is a method of determining a patient's risk for PML. Themethod includes determining the patient's previous exposure to ananti-VLA-4 therapy, and determining whether the patient previouslyreceived treatment with an immunosuppressant. Optionally, the patient'santi-JCV antibody status may also be determined. If the patient isdetermined to have received an anti-VLA-4 therapy for 24 months or less,and not to have previously received treatment with an immunosuppressant,then the patient is determined to be at lower risk for PML. If thepatient is determined to have received anti-VLA-4 therapy for longerthan 24 months, and not to have previously received treatment with animmunosuppressant, then the patient is determined to be at higher riskfor PML. A patient determined to be at lower risk for PML may further beadministered an anti-VLA-4 therapy, e.g., natalizumab. Conversely, apatient determined to be at higher risk for PML may further beadministered an alternative to anti-VLA-4 therapy, e.g., an interferon,a corticosteroid, a statin or a TNF antagonist.

In one embodiment, the patient's JCV status is also determined, and ifthe patient is determined to be JCV negative, then the patient isdetermined to be at a lower risk for PML than if the patient wasdetermined to be JCV positive.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B are graphs depicting natalizumab-associated PMLincidence by cumulative treatment duration (FIG. 1A) and by 12-monthtreatment interval duration (FIG. 1B).

FIG. 2 is a schematic diagram depicting the approximate incidence of PMLstratified by prior immunosuppressant use and natalizumab treatmentduration.

FIG. 3 is a schematic diagram depicting the approximate incidence of PMLstratified by anti-JCV antibody sero status, prior immunosuppressantuse, and natalizumab treatment duration.

FIGS. 4A and 4B are graphs depicting sensitivity analyses of PMLincidence estimates in anti-JCV antibody positive patients, stratifiedby prior immunosuppressant use (yes or no) and natalizumab treatmentduration (1-24 months (FIG. 4A) or 25-48 months (FIG. 4B)). Base=Basecase scenario.

FIGS. 5A and 5B are graphs depicting nODs and titers, respectively, ofpatient 1.

FIGS. 6A and 6B are graphs depicting nODs and titers, respectively, ofpatient 2.

FIGS. 7A and 7B are graphs depicting nODs and titers, respectively, ofpatient 3.

FIGS. 8A and 8B are graphs depicting nODs and titers, respectively, ofpatient 4.

FIGS. 9A and 9B are graphs depicting nODs and titers, respectively, ofpatient 5.

FIGS. 9C and 9D are graphs depicting nODs and titers, respectively, ofpatient 6.

FIGS. 10A and 10B are graphs depicting nODs and titers, respectively, ofpatient 7.

FIG. 11 is a scatter plot depicting index (x axis) and percentinhibition (y axis) data collected for a group of MS patients.

FIG. 12 is a scatter plot depicting index (x axis) and percentinhibition (y axis) data collected for a group of MS patients.

FIG. 13 is a scatter plot depicting index values determined for a groupof MS patients.

DETAILED DESCRIPTION

The invention is based, at least in part, on the discovery of new andimproved methods of assessing the risk of a patient for PML that includeassessing anti-JCV antibody titers or percent inhibition. The inventionis based at least in part on the discovery that anti-JCV antibody titerand percent antibody inhibition can be an indicator of a patient's riskof developing Progressive Multifocal Leukoencephalopathy (PML).

Applicants have also developed an optimized assay for determininganti-JCV antibody titer levels in a biological sample, and a method forassaying the antibodies qualitatively by determining percent inhibitionvalues, and using this information to determine the risk of a patientfor developing PML. The assay includes: (a) forming a first reactionmixture comprising a first aliquot of a sample and a substrate on whichis disposed HPVLPs, where, the VLP particles are present at an amount of0.04 μg, and a concentration of 0.4 μg/mL; b) detecting the level ofanti-JCV antibody bound to HPVLP disposed on the substrate, such as bydetecting a labeled secondary detection reagent, e.g., an enzyme labeledanti-IgG antibody, bound to anti-JCV antibody bound to said substrate;(c) forming a second reaction mixture comprising a second aliquot ofsample with solution-phase HPVLP provided at a concentration of, e.g.,0.4 μg/mL, and a second aliquot of sample provided at, e.g., a 1:100 or1:101, dilution; (d) forming a third reaction mixture comprising anegative control solution containing no HPVLP, and a third aliquot ofsample diluted, e.g., 1:100 or 1:101, or 1:110 in the negative controlsolution; (e) detecting the level of unbound anti-JCV antibody in thesecond and third reaction mixtures, such as by detecting JCV capable ofbinding a substrate on which is disposed HPVLPs, where said HPVLP ispresent; (f) providing a first value, which corresponds to the level ofanti-JCV antibody binding to HPVLP disposed on substrate in the firstaliquot of sample, and a second value, which corresponds to the level ofunbound anti-JCV antibody in the second reaction mixture, e.g., thelevel anti-JCV antibody that binds to HPVLP disposed on a substrate fromsaid second reaction mixture; and (g) optionally, comparing the firstand second antibody levels.

Applicants have also discovered that a patient has a lower risk ofdeveloping PML if, (i) the anti-JCV antibody titer as indicated by indexvalue or nOD is determined to be <0.5, or (ii) the anti-JCV antibodytiter as indicated by index value or nOD is determined to be >0.5 and<3.0, and the percent inhibition is determined to be less than or equalto 70%. The patient has a higher risk of PML if, (i) the anti-JCVantibody titer as indicated by index value or nOD is determined to be >3and the percent inhibition value is determined to be >70%, or (ii) thepatient showed an increase in index, nOD or titer by 2-fold from aprevious test.

A patient can be monitored at regular intervals, such as every 6 monthsor every 12 months for a change in anti-JCV antibody titer or percentinhibition. If the results of this later assay indicate that the patientstill has an anti-JCV antibody titer of nOD less than 0.5, and a percentinhibition of <70%, then the patient can be determined to still be at alower risk for developing PML. If a later assay indicates that thepatient's antibody titer is increased by 2 to 3 fold from the initialassay, then the patient can be determined to be at increased or higherrisk for developing PML. Applicants observed patients diagnosed with PMLtend to demonstrate an increase in antibody titer and nOD by 2 to 3 foldin the six months prior to diagnosis.

A patient has a higher risk of PML if, (i) the anti-JCV antibody titeras indicated by index value or nOD is determined to be >3 and thepercent inhibition value is determined to be >70%, or (ii) the patientshowed an increase in index, nOD or titer by 2-fold from a previoustest.

A patient satisfying these criteria can, optionally, be determined notto be a candidate to receive therapy with an anti-VLA-4 therapy, such asan anti-VLA-4 antibody, e.g., natalizumab, or the patient can further beassessed for other risk factors of developing PML. These risk factorsinclude whether or not the patient has previously received an anti-VLA-4therapy, such as natalizumab, and for how long the patient has receivedthe therapy; and whether and for how long the patient has previouslyreceived an immunosuppressant therapy other than an anti-VLA-4 therapy.A patient's risk of PML is a combination of each of these factors.

Antibody titer can be measured by “nOD” or “index.” “nOD” is thenormalized optical density value in a test, such as an ELISA test, foranti-JCV antibody detection. The “index” value is the optical densityvalue for the sample divided by the optical density of the positivecontrol in an immunoassay, such as the ELISA assay.

Applicants previously discovered that patients who received ananti-VLA-4 therapy, such as natalizumab, for 24 months or less, and whohave not previously received an immunosuppressant therapy, are at lowerrisk for developing PML, than patients who do not meet these twocriteria. Further, patients who have the lowest risk are those who meetthese two criteria, and who are also JCV negative, e.g., patients who donot test positive for anti-JCV antibodies or JCV nucleic acid, e.g., JCVDNA. It was previously unknown that each of these three risk factors((i) the amount of time the patient has previously received ananti-VLA-4 therapy; (ii) whether or not a patient has previouslyreceived treatment with an immunosuppressant other than an anti-VLA-4therapy; and (iii) JCV status) independently contribute to a patient'srisk of PML. The inventions described herein can be used in general forpatients treated with a VLA-4 inhibitor. The ability to identifysubpopulations of patients at distinctly different PML risks allows forbetter characterization of risk than previous methods (i.e., overall PMLrisk) and should assist healthcare professionals and patients in makingmore informed benefit-risk treatment decisions. These risk assessmentcriteria are described in co-owned U.S. provisional applications61/491,810, filed May 31, 2011, and 61/508,584, filed Jul. 15, 2011. Thecontents of each of these provisional applications is herebyincorporated by reference in its entirety. The risk criteria describedherein directed to anti-JCV antibody titer (e.g., as measured by nOD orindex level) and percent inhibition can be considered in combinationwith the risk factors described in the prior co-owned provisionalapplications.

The methods for determining PML risk can require acquiring one, two orall three of a JCV classification for a patient (e.g., anti-JCV antibodytiter, such as measured by nOD or index level and percent inhibition),prior anti-VLA-4 therapy history for the patient, and priorimmunosuppressant therapy history (other than anti-VLA-4 therapy) forthe patient. Responsive to these classifications, a patient can beassigned a treatment suitability classification. Patients who aredetermined to have low risk of developing PML can be assigned a positivetreatment classification, and patients who are determined to have ahigher relative risk of developing PML can be assigned a negativetreatment classification. A patient who receives a positive treatmentclassification can receive a recommendation for further treatment or forinitiating treatment with an anti-VLA-4 therapy. A patient receiving anegative treatment classification may receive a recommendation toterminate treatment with an anti-VLA-4, a recommendation to initiatetreatment with a non-anti-VLA-4 therapy, a recommendation for continuingor initiating anti-VLA4 therapy with increased surveillance for signsand symptoms of PML.

A recommendation for further treatment with an anti-VLA-4 therapy may beaccompanied with further instructions or requirements that the patientreceive additional or enhanced monitoring, such as if one or morefactors indicate that the patient may be at an increased risk of PML,e.g., prior treatment with an anti-VLA-4 therapy for longer than 24months, e.g., 25 months or longer, or prior treatment with animmunosuppressant other than an anti-VLA-4 therapy.

A patient can be determined to have previously received an anti-VLA-4therapy or an immunosuppressant therapy other than an anti-VLA-4 therapythrough self-reporting by the patient, or through information (verbal orwritten) provided by a parent, physician, physician's assistant, nurseor other healthcare provider. The information can also be obtainedthrough a database, such as a medical database or a clinical trialsdatabase.

Prior immunosuppressant therapies, other than anti-VLA-4 therapy, thatwill be indicative of an increased risk of PML can include priortreatment with antineoplastics, immunosuppressants or immunomodulators,such as one or more beta-interferon or glatiramer acetate. Exemplaryimmunosuppressants include, e.g., mitoxantrone, methotrexate,azathioprine, cyclophosphamide, and mycophenolate, anti-CD20 therapy(e.g., rituximab), an anti-CD11a therapy (e.g., efalizumab), ormycophenolate mofetil. Prior treatment with other immunosuppressanttherapies as described below will also be predicted to increase apatient's risk of PML following further administration of an anti-VLA-4therapy. In general, a determination of prior immunosuppressant use is aspecified use which can be any prior use of an immunosuppressant that isnot a VLA-4 inhibitor (e.g., an anti-VLA-4 antibody) or prior use withina specified period of time, for example, within the previous 1, 2, 3, 5,or 10 years prior to the evaluation of PML risk.

Determining JCV status refers to determining whether a patient has beenexposed to JCV and therefore includes direct methods of determiningexposure (for example, detecting JCV proteins or JCV DNA) and indirectmethods (e.g., detecting antibodies against JCV in a patient sample).Assays for determining JCV status can include assays for detecting JCVnucleic acid (e.g., DNA or RNA), or JCV seroprevalence, or anti-JCVantibodies in a biological sample, such as in plasma, serum, blood orurine sample, or in a sample of peripheral blood mononuclear cells(PBMCs), or cerebrospinal fluid. JCV nucleic acid can be detected usingmethods known in the art, for example, by an amplification method, e.g.,polymerase chain reaction (PCR), or by a Next Generation Sequencing(NGS) method. JCV seroprevalence can be assayed using methods known inthe art such as a haemagglutination inhibition (HI) assay. JCVantibodies can be detected by an immunoassay, such as an ELISA assay. Inone embodiment, JCV antibodies can be detected by the method describedin International Application Number PCT/US2011/20832, which utilizesHPVLPs under conditions suitable for binding of an anti-JCV antibody fordetecting the level of anti-JCV antibody binding in a biological sample.Methods of determining JCV status also include methods of determininganti-JCV antibody titer and percent inhibition. Detection of anti-JCVantibody titer and percent inhibition typically include a two-stepantibody detection assay as described in International ApplicationNumber PCT/US2011/20832.

If the presence of JCV is identified in a biological sample from apatient, e.g., JCV antibodies, proteins, peptides, or nucleic acids, thepatient is determined to be “JCV positive.” A positive JCVclassification corresponds to the presence of JCV antibodies in thebiological sample, e.g., JCV antibodies that are equal to or greaterthan a preselected criterion. The preselected criterion is typically aqualitative value, e.g., a “detectable” amount of antibody according toa particular assay, e.g., an immunoassay.

The methods described herein for determining PML risk can be useful forany human subject, including a subject considering treatment with animmunomodulator, for example an anti-VLA-4 therapy (e.g., natalizumab),an anti-CD20 therapy (e.g., rituximab), an anti-CD11a therapy (e.g.,efalizumab), or mycophenolate mofetil; in a subject currently beingtreated with an immunomodulator; or a subject that has ceased treatmentwith an immunomodulator. The method may be useful to others who may besusceptible to PML such as individuals having lymphoproliferativedisorders, such as multiple myeloma or a lymphoma; individuals infectedwith human immunodeficiency virus (HIV), or having acquired immunedeficiency syndrome (AIDS), hematologic malignancies, or an autoimmunedisease such as systemic lupus erythematosus (SLE), an inflammatorybowel disease, such as Crohn's Disease (CD) or ulcerative colitis,multiple sclerosis (MS) or arthritis, e.g., rheumatoid arthritis (RA).The risk-assessment method may also be useful to subjects receivingimmunosuppressive or immunomodulatory therapies, such as transplantpatients. Exemplary immunosuppressive or immunomodulatory therapiesinclude natalizumab, rituximab, efalizumab, and mycophenolate mofetil.The method can be useful for assessing risk in a subject having adisorder, or being treated with a drug, disclosed in Piccinni et al.“Stronger association of drug-induced progressive multifocalleukoencephalopathy (PML) with biological immunomodulating agents” Eur.J. Clin. Pharmacol. 66:199-206, 2010, the contents of which areincorporated herein by reference.

Definitions

As used herein, an “HPVLP” is a highly purified VLP (“virus-likeparticle”) consisting predominantly of the VP1 protein. An “HPVLP”featured in the invention is composed mainly of the major capsid protein“VP1,” which can be a naturally-occurring VP1 or a recombinant VP1, fromthe polyomavirus, JC Virus (JCV). An HPVLP can be composed of, e.g., atleast one pentameric subunit, more than one pentameric subunit, up toseventy-two pentameric subunits or more of VP1. An HPVLP of theinvention can bind antibodies against naturally-occurring, intact JCvirus. In some embodiments, an HPVLP includes a second, and optionally athird, polypeptide that is a minor capsid protein of JC virus, e.g., atleast one VP2 or VP3 polypeptide. The VP2 or VP3 can be recombinant ornaturally-occurring or naturally-derived polypeptides.

Such “highly purified” particles contain more than one VP1 pentamer,e.g., at least 5, 10, 20, 30, 40, 50, 60, 70, 72 VP1 pentamers, or lessthan 100 VP1 pentamers. Such highly purified particles can be obtained,for example, by a method that involves double filtration. For example,in one embodiment, a highly purified preparation of VLPs is obtained bypurifying the particles at least twice by centrifugation, e.g., througha sucrose cushion. In other embodiments, HPVLPs are prepared usingchromatographic methods. In general, an HPVLP preparation can beidentified by its activity in an ELISA assay using defined controlsamples. In some cases, such control samples are negative controlsand/or control samples containing low levels of JCV antibodies.

As used herein, a “high signal-to-noise HPVLP substrate” is a substrateon which is disposed HPVLP. It can be used to evaluate the level of free(that is unbound to antigen or other target, e.g., HPVLP, in a sample.The concentration of HPVLP on the substrate is such that, when measuringthe amount of anti-JCV antibody present, it provides forasignal-to-noise ratio of 10 to 30, 15 to 30, 15 to 25, 18 to 22. Inembodiment the signal-to-noise ratio is at least 10, 15, 18 or 20. Inembodiments signal-to-noise ratio is about 10, 15, 18 or 20. Thesignal-to noise ratio can be determined with a sample, e.g., acalibration control, that gives an optical density of 1.0. In anembodiment the HPVLP is provided on said substrate at a concentrationwhich results from lyophilizing 0.5 ml, 0.8 ml, 1.0 ml, 1.2 ml, or 1.5ml of 0.4 μg/ml of HPVLP in a well of a 96 well plate. In an embodimentthe HPVLP is provided on said substrate at a concentration which resultsfrom lyophilizing 1.0 ml of 0.4p g/ml of HPVLP in a well of a 96 wellplate, which as used herein, is equivalent to 30 ng to 50 ng (e.g., 40ng) HPVLP per well. In an embodiment the HPVLP is provided on saidsubstrate at a concentration which results from lyophilizing 0.05 ml to0.35 mL or 0.1 ml to 0.2 ml of 0.4 μg/ml of HPVLP in a well of a 96 wellplate. The amount of HPVLP disposed on the substrate, or the conditionsunder which deposition is achieved, can vary as long as the desiredsignal-to-noise ratio is obtained.

A signal-to-noise ratio is computed by comparing the optical densityvalue of the negative control to the calibrator control to determine thedynamic range of the signal intensity in the assay.

In an embodiment the sample is diluted about 100 fold and the cut offfor negative score is a reduction that is less than or equal to 45% and,the cutoff for a positive score is greater than 45%. In embodiments thedilution is other than 100 fold but is less than 200 fold. For example,the dilution is between 50- and 150-fold, 75- and 125-fold, 85- and115-fold. In embodiments, the dilution is less than 150-fold, 125-fold,100-fold, or 75-fold. In embodiments where the dilution is other than100-fold (e.g., 200-fold 400-fold, 500-fold, 800-fold, upto >1,000,000-fold, the cutoff, or other parameters, are adjusted suchthat a sample would receive the same score (positive or negative) as itwould if the dilution was 100-fold and the cut off for negative is lessthan 45% and the cut off for positive is greater than or equal to 45%.

Anti-JCV Antibody Detection Assay. Assays are conducted by adding abiological sample to a substrate that has been coated with an HPVLP anddetected using methods known in the art. In general, a solid baseplatform is used such as a microtiter plate (for example, a 96 wellplate); although other formats known in the art can be used. In someembodiments, the biological sample is diluted prior to use in an assay.

In certain embodiments, the assay format is an enzyme-linked immunoassay(ELISA). Broadly, the method typically includes coating the substratewith capture antigen such as HPVLP, incubating sample containing bindingantibodies directed to capture reagent, washing to removenon-specifically bound species, and detecting the bound immunecomplexes, e.g., by a chromogenic or chemiluminescent assay. Chromogenicsubstrates produce a colored end product, which can be detected andmeasured visually or with the use of a spectrophotometer.Chemiluminescent substrates produce light, which can be measured using aluminometer.

Coating a plate with HPVLP generally includes incubating the solidsubstrate (such as wells of a microtiter plate) with a solution of HPVLPat a suitable concentration (e.g., 0.4 μg/ml), either overnight or for aspecified number of hours. The HPVLP can include VP1 as the only JCVviral component, or the HPVLP can be a heterologous particle, thatcontains at least one of VP2 or VP3 per particle or at least one each ofVP2 and VP3 per particle. After coating with the HPVLP, the wells of theplate are washed. The substrate is then “coated” with a nonspecificprotein that is antigenically neutral with regard to the samples to betested. Suitable coating materials are known in the art and includebovine serum albumin (BSA), casein, sugars or solutions of milk powder.Plates may then be dried and stored for a longer period of time, such as1 days, 1 month or 1 year prior to proceeding to the next step of theassay.

The sample or reference is incubated on the prepared substrate underconditions effective to permit complex formation (HPVLP/JCV antibody),thus forming a bound complex. Detection of the bound complex isperformed using a labeled antibody that can bind to human antibody. Ingeneral, the labeled antibody can detect human IgG or human IgG and IgM.In some cases, the assay can be performed using secondary or tertiarydetection methods.

A reference sample can be of the same biological material (e.g., plasma,serum, urine, or CSF) isolated from an individual known to be infectedwith JC virus based on the presence of JCV DNA in urine of theindividual (uropositive). A reference sample is used to establish theassay cut-point such that the false negative rate of the assay is notgreater than 1%-3%.

“Under conditions effective to permit complex formation” generally meansconditions in which the reagents have been diluted to reduce backgroundand provide readouts of results that lie within a specified range.Diluents can include, in non-limiting examples, solutions that includeBSA, phosphate buffered saline (PBS), or PBS containing Tween.

“Suitable” conditions also include conditions that are at a temperatureand/or for a period of time sufficient to allow effective binding.Incubations are typically from about one to two hours or one to fourhours, at temperatures of approximately 25° C. to 27° C., or may beovernight at about 4° C. However, those in the art will understand thatother conditions may be suitable.

In general, one or more washes are conducted between the incubations ofthe assay. Appropriate wash solutions include diluent buffer (e.g., PBSor PBS/Tween) or borate buffer.

In general, the detection of antibody bound to HPVLP is performed usingmethods well known in the art. In general, such methods are based on thedetection of a label or marker, such as a radioactive, fluorescent,biological or enzymatic tag. U.S. patents concerning the use of suchlabels include, for example, U.S. Pat. Nos. 3,817,837; 3,850,752;3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. In general,the detection of anti-JCV antibody binding is detected using a secondaryantibody that is labeled. In general, the secondary antibody is specificfor detecting human IgG. Quantification is achieved by measuring thedegree of color generated, e.g., using a visible spectraspectrophotometer.

In one embodiment, the assay is performed in a medical office, such asby a healthcare provider, e.g., a doctor, a nurse or a technician,working in a facility where the biological sample is obtained from apatient. In another embodiment, the biological sample obtained from apatient is transported to another facility, e.g., to a third partyfacility, where the assay is performed. In this latter case, the resultsof the assay can be reported back to the healthcare provider, such asthrough a form, which can be submitted by mail or electronically (e.g.,through facsimile or e-mail) or through an on-line database. In oneembodiment, the results of the assay (including the screening assay and,optionally, a confirmatory assay) can be stored in a database and can beaccessed by a healthcare provider, such as through the worldwide web.

Secondary Test. In some cases, for example, when the level of anti-JCVantibody in a sample falls into a designated “equivocal zone” or“indeterminate zone,” e.g., where it is determined that there is limitedcertainty regarding the presence or absence of anti-JCV antibody (suchas when the nOD value is determined to be >0.2 and <0.4), a secondarytest (also referred to herein as a “confirmatory assay”) of the sampleis employed. For the secondary test, two aliquots of a biological sampleare used. The first is prepared prior to use in the assay bypreincubating the sample in the presence of assay buffer in solution fora period of time (e.g., for 30 minutes, one hour, or longer such asovernight at 4° C.). The second aliquot is prepared prior to use in theassay by preincubating the sample in the presence of HPVLP in solutionfor a period of time (e.g., for 30 minutes, or one hour or longer). Thetwo aliquots are then used in the HPVLP assay as described herein, andthe assignment of the sample to anti-JCV antibody positive or antibodynegative is made. If the assay results for the aliquot incubated withHPVLP indicate a value of <45% inhibition (i.e., the “cut-point”), thenthe sample is interpreted to be negative for the presence ofJCV-specific antibodies. If the assay results indicate a value of ≥45%inhibition, then the sample is interpreted to have JCV-specificantibodies and therefore as antibody positive.

An assay featured in the invention that utilizes a secondary test isalso referred to herein as a “two-step test” or a “two-step assay.” Anearlier version of the two step assay is described in co-ownedInternational Application No. PCT/US2071/020832, which is incorporatedby reference herein in its entirety.

Substrates' and Solution Based Methods. Any suitable solid substrate canbe used for the HPVLP assay format. In some embodiments, the substrateis a microtiter plate (e.g., a 96-well plate) a slide, a bead, or acolumn. The substrate can be suitable for chromogenic orchemiluminescent detection methods, or solution based methods such asproximal ligation.

Cut-point. The invention provides methods of analysis that employ“cut-points” to reduce false negative and false positive rates. Thecut-points are established based on data from the HPVLP assays (e.g., todetect JCV antibodies in a biological sample), averaged, for example,between duplicate test samples and multiple replicates (for example, atleast two, at least four, or at least eight replicates of controlsamples). Cut-points can also be determined statistically using largepanels of non-PML and PML samples.

In one version of an assay according to the present invention, resultsfrom initial HPVLP screening assays, e.g., ELISA assays, will cause atest sample to be classified as having or not having JCV-specificantibodies, or, if the sample does not fall under one of these twoclassifications, then the sample will be subjected to a supplementalconfirmation assay. For example, samples that produce a result in anHPVLP ELISA assay featured in the invention less than an establishedlevel (e.g., an nOD₄₅₀<0.2) will be classified as lacking JCV-specificantibodies, and samples that provide a result in the ELISA greater thanan established level (e.g., an nOD₄₅₀>0.4) will be classified aspositive for JCV-specific antibodies. Samples that do not clearly fallinto one of these classifications (e.g., 0.2<OD₄₅₀<0.4) can be tested ina confirmatory assay.

In one embodiment, the confirmatory assay requires a pre-incubationstep, where the test sample is pre-incubated with buffer (or othersuitable solution) control or with HPVLPs (in buffer or other suitablesolution) to pre-adsorb JCV-specific antibodies prior to analysis in anHPVLP ELISA, as described in further detail below. After pre-incubationwith HPVLP if the reaction in the primary assay decreases by less than45% compared to buffer control, then the sample is interpreted to benegative for the presence of JCV-specific antibodies. If the resultsshow a ≥45% reduction in reaction compared to buffer control in theprimary assay after pre-incubation with HPVLP then the sample isinterpreted to contain JCV specific antibodies. In some embodiments,only the confirmatory assay is performed.

VP1. The use of HPVLPs in an assay for JCV antibodies can improve theaccuracy of the assay and is useful in an assay suitable for analyticand diagnostic purposes. VP1 for use in producing HPVLPs can begenerated using methods known in the art and can be eithernaturally-occurring VP1 or recombinantly produced VP1, e.g., a VP1 froma JC virus. In general, the VP1 used is VP1 from a MAD1 strain of JCV.In some embodiments, the VP1 used in the assay comprises VP1 from morethan one JCV strain, for example, from one or more of strains 1A, 1B,2A, 2B, 3, 4, and 7. After preparation of VP1, e.g., recombinantlysynthesized VP1, the VP1 for use in the assays described herein is thenfurther purified through standard biochemical methods includingdensity-gradient/ultracentrifugation methods, or a series of chemicalprecipitation steps, concentration/diafiltration and ion-exchangechromatography. The purification methods typically include a step toremove smaller proteins including monomer VP1 polypeptides, or pentamerVP1. The removal of these smaller particles can be done in, for example,in one step or in two steps (e.g., a first filtration step to remove VP1monomers, and then a second filtration step to remove pentamer VP1particles). Such biochemical purification methods are known to those inthe art. Examples 1 and 7 provide two different methods of JCV VP1-VLPpurification.

An HPVLP preparation (HPVLPs) according to one aspect of the presentinvention does not contain significant amounts of VP1 monomer (e.g., hasbeen purified to remove monomers). An HPVLP preparation according toanother aspect of the present invention does not contain significantamounts of VP1 molecules in configurations the size of a VP1 pentamer,or smaller (including monomer). The HPVLP can be prepared fromrecombinant VP1 or naturally-occurring VP1 (e.g., isolated from virus orvirus capsid). In some embodiments, additional JCV components, such asone or both of the minor coat proteins from JC virus, e.g., VP2 or VP3,are included in the HPVLP particle or are associated with the substrate.

In some cases, recombinantly expressed VP1 may not assemble intopentamers or HPVLPs that resemble naturally-occurring viral capsids, forexample, recombinantly expressed VP1 may assemble into tubes or othernon-spherical geometries. Accordingly, the invention relates to methodsof producing HPVLPs that are substantially spherical in geometry. Theinvention includes HPVLP preparations where at least about 10%, about15%, about 20%, about 25%, about 50%, about 60%, about 65%, about 70%,about 80%, about 90%, about 95%, or about 99% of the HPVLPs in thepreparation resemble the naturally-occurring JCV capsid (e.g., are in anicosahedral or substantially spherical configuration). In someembodiments, an HPVLP preparation contains at least 10%, at least 15%,at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 99% of the HPVLPs in thepreparation resemble the naturally-occurring JCV capsid. Such methodscan include expressing viral proteins under conditions that result insuch a preparation and/or isolating and purifying expressed viralproteins as described herein to produce such a preparation.

Methods of Making HPVLPs. HPVLPs can be made, for example, bytransforming a baculovirus with a vector expressing a VP1 gene, such asa VP1 gene from a JC virus. The baculovirus is used to infect a cellculture, such as an insect cell culture (e.g., SF9 cells) or a mammaliancell culture, and the cells express the VP1 protein. HPVLPs are isolatedby lysing the cells, and purifying the particles through a series ofcentrifugation and ultrafiltration steps. In general, the purificationis performed using methods such as sucrose cushion sedimentation,isopycnic ultracentrifugation and extensive ultrafiltration or othermethods known to those in the art. In certain embodiments, thepurification will include twice centrifuging the particles through asucrose cushion. In an alternative purification method, cells are lysed,and particles are isolated by a series of precipitation andconcentration/diafiltration steps with a final ion-exchange step. In yetanother alternative method, the HPVLPs are purified by chromatographicmethods, and without centrifugation steps.

Purity can be assessed using any suitable techniques known in the art,for example, analytical ultracentrifugation, electron microscopy, PAGEanalysis, mass spectrometry, protein concentration, or activity in anELISA with control sera.

Insufficiently purified VLPs result in a high background yieldingfalsely high anti-JCV antibody levels or calculated exposure rates.

In some embodiments, the HPVLPs contain VP1 as the sole JC virusprotein.

In some embodiments, the HPVLPs are heterogeneous particles, andtherefore include VP1 protein, and at least one of the minor coatproteins of JC virus, e.g., VP2 or VP3. In another embodiment, the HPVLPincludes VP1, VP2 and VP3 proteins. An HPVLP that includes VP1 and VP2can be produced using methods known in the art, for example, bytransforming a baculovirus with a nucleic acid including a VP1 and a VP2gene, such as under the control of the same or different promoters. Acell culture is infected with the baculovirus, and the cells express VP1and VP2, and HPVLPs form which include both types of proteins. In oneembodiment, the VP1 and VP2 genes are on different DNA molecules, theDNA molecules are transformed into different baculoviruses and thebaculoviruses are used to transfect cells in the same culture. The cellsexpress the VP1 and VP2 proteins, and HPVLPs form which include bothtypes of protein. In some cases, a heterogeneous HPVLP will include,e.g, one or two VP2 polypeptides for every five VP1 polypeptides. Ingeneral, an HPVLP will contain more VP1 polypeptides than VP2polypeptides, as is the case in naturally-occurring JC virus.

An HPVLP that includes both VP1 and VP3 or both VP1 and VP2 moleculescan be produced, for example, by transforming a baculovirus with anucleic acid including a VP1 and a VP3 gene or a VP1 and VP2 gene,respectively, under the control of the same or different promoters. Acell culture is infected with the baculovirus, and the cells express VP1and VP3 or VP1 and VP2, and HPVLPs form which include both types ofproteins. In some embodiments, the VP1 and VP3 or VP1 and VP2 genes areon different DNA molecules, the DNA molecules are transformed intodifferent baculoviruses, and the baculoviruses are used to transfectcells in the same culture. The cells express the VP1 and VP3 proteins orVP1 and VP2 genes, respectively, and HPVLPs form which include bothtypes of protein. HPVLP particles can be isolated from such preparationsusing methods known in the art such as those used to isolate JCVcapsids.

Typically, a VP1 pentamer that is in a heterogeneous HPVLP will include,e.g, five VP1 polypeptides and one VP3 polypeptide and/or one VP2polypeptide, depending on whether a VP3 gene or VP2 gene was used tomake the constructs. There will typically be more VP1 polypeptides thanVP3 or VP2 polypeptides in an HPVLP. In some embodiments, the VP2 or VP3is from a polyoma virus that is not a JC virus, e.g., a BK viruspolypeptide.

An HPVLP that includes all three of VP1 and VP2 and VP3 molecules can beproduced by transforming a baculovirus with a nucleic acid (e.g., acircular DNA, e.g., <5.5 kb) including a VP1, VP2 and VP3 gene, such asunder the control of the same or different promoters. A cell culture,such as a mammalian cell culture, is infected with the baculovirus, andthe cells express VP1, VP2 and VP3 proteins. HPVLPs consequently formwhich include all three types of proteins. In one embodiment, the VP1,and either or both of the VP2 and VP3 genes are on different DNAmolecules, the DNA molecules are transformed into the same or differentbaculovirus, and the baculovirus are used to infect cells in the same orseparate cultures. The cells express the VP1, VP2 and VP3 proteins, andHPVLPs form which include both types of protein. A heterogeneous HPVLPcan include, e.g, five VP1 polypeptides and one each of VP2 and VP3polypeptides, although the ratios may vary within a preparation. Therewill typically be more VP1 polypeptides than VP2 and VP3 polypeptides inan HPVLP.

In some embodiments, the HPVLP will be greater in size than a VP1pentamer. By greater in size, it is meant that the mass of proteincontained in an HPVLP particle is greater than a pentamer containingsolely VP1.

In other embodiments, the method of preparing a solution of HPVLP caninclude removing from the solution particles (e.g., VP1 monomers orsmall VP1 containing particles) that are the size of a VP1 pentamer orsmaller. Methods such as centrifugation and size-exclusionchromatography can be used to perform this purification step. In someembodiments, other methods known in the art, e.g., ion exchangechromatography, can be used in the preparation of HPVLPs that are largerthan a VP1 pentamer. In general, an HPVLP preparation suitable for usein an assay will contain at least 20% HPVLPs, at least 25% HPVLPs, atleast 40% HPVLPs, at least 60% HPVLPs, at least 65% HPVLPs, at least 70%HPVLPs, at least 80% HPVLPs, at least 85% HPVLPs, at least 90% HPVLPs,at least 95% HPVLPs, or at least 99% HPVPLs compared to non-HLVLPparticles (e.g., by percent of pentamers compared to VP1 monomers andaggregates containing fewer than five VP1 molecules).

Methods of Evaluating Samples and/or Subjects. As used herein, methodsof evaluating or analyzing a subject or biological sample from a subjectinclude one or more of performing the analysis of the sample, requestinganalysis of the sample, requesting results from analysis of the sample,or receiving the results from analysis of the sample. (Generally herein,determination (or determining), analysis or evaluation (or evaluating)can include one or both of performing the underlying method or receivingdata from another who has performed the underlying method.)

The analysis or evaluation requires a transformation of material, e.g.,biological material or assay components. For example, a biologicalsample can be evaluated for the presence of anti-JCV antibodies,anti-JCV antibody titer and percent inhibition of JCV antibodies. Theevaluation can be performed before or after or at the same time thepatient is receiving treatment, such as for MS. The evaluation is based,at least in part, on analysis of a sample from the subject, e.g., ablood, plasma, serum, urine or CSF sample. The presence of anti-JCVantibodies can be determined by contact with a specific binding agent,e.g., a JCV protein, such as VP1. The binding agent can be a JCVprotein, e.g., VP1 in the form of a particle, e.g., a HPVLP.

In one embodiment, an assay to detect the presence of anti-JCVantibodies is a two-step assay, such as described herein. The assayutilizes HPVLPs under conditions suitable for binding an anti-JCVantibody. The assay is capable of detecting any isotype of anti-JCVantibody (including IgG, IgM, IgA, and IgE). The assay is also highlysensitive and can detect anti-JCV antibodies at a concentration of, forexample, 2.0 μg/mL or less, e.g. 1.5 μg/mL or less, 1.25 μg/mL or less,1.0 μg/mL or less, 0.5 μg/mL or less, 50 ng/mL or less, 10 ng/mL orless, 5 ng/mL or less, 1.7 ng/mL or less, or 1 ng/mL or less.

In one embodiment, the sample is analyzed for the level of JCV nucleicacid present in the sample. For example, nucleic acids can be isolatedfrom the sample and used for PCR amplification or a Next-Generation(Nex-Gen) Sequencing technique. In one embodiment, a crude lysate of thebiological sample is subject to an amplification method, such as PCR,and the amplified product is analyzed by one or more of electrophoresis,restriction fragment mapping, hybridization or sequencing to identifywhether JCV DNA or RNA is present in the sample and how much is in thesample.

The biological sample can be removed from the patient and analyzed.

In some embodiments, the patient sample, e.g., a serum or plasma orwhole blood sample or CSF, can be stored prior to testing for JCV, e.g.,for JCV antibodies or for JCV nucleic acid. The patient sample, e.g.,the patient sample containing JCV antibodies or JCV nucleic acid, can bestored for 1-21 days, e.g., 1-14 days or 1-7 days or longer (e.g., oneday, two days, three days, five days, seven days, ten days, 14 days, 21days or longer); for one to six weeks, e.g., one to three weeks or oneto two weeks or longer (e.g., up to one week, up to two weeks, up tothree weeks, up to six weeks, or longer); or for one to six months,e.g., one to three months or one to two months or longer (e.g., up toone month, up to two months, up to three months, up to six months orlonger). The sample can be stored, for example, frozen (e.g., at −80° C.to −20° C.), at 2-8° C., at ambient temperature (18° C.-25° C.) orwarmer, e.g., at 37° C.

As used herein, the term “acquire” or “acquiring” refers to obtainingpossession of a physical entity, or a value, e.g., a numerical value, by“directly acquiring” or “indirectly acquiring” the physical entity orvalue, e.g., the status of a patient, such as prior exposure toanti-VLA-4 therapy or other immunosuppressants, or JCV status. “Directlyacquiring” means performing a process (e.g., performing a synthetic oranalytical method) to obtain the physical entity or value. “Indirectlyacquiring” refers to receiving the physical entity or value from anotherparty or source (e.g., a third party laboratory that directly acquiredthe physical entity or value). Directly acquiring a physical entityincludes performing a process that includes a physical change in aphysical substance, e.g., a starting material. Exemplary changes includemaking a physical entity from two or more starting materials, shearingor fragmenting a substance, separating or purifying a substance,combining two or more separate entities into a mixture, performing achemical reaction that includes breaking or forming a covalent ornon-covalent bond. Directly acquiring a value includes performing aprocess that includes a physical change in a sample or anothersubstance, e.g., performing an analytical process which includes aphysical change in a substance, e.g., a sample, analyte, or reagent(sometimes referred to herein as “physical analysis”), performing ananalytical method, e.g., a method which includes one or more of thefollowing: separating or purifying a substance, e.g., an analyte, or afragment or other derivative thereof, from another substance; combiningan analyte, or fragment or other derivative thereof, with anothersubstance, e.g., a buffer, solvent, or reactant; or changing thestructure of an analyte, or a fragment or other derivative thereof,e.g., by breaking or forming a covalent or non covalent bond, between afirst and a second atom of the analyte; or by changing the structure ofa reagent, or a fragment or other derivative thereof, e.g., by breakingor forming a covalent or non covalent bond, between a first and a secondatom of the reagent.

At least one or both of determining a patient's status (e.g., JCVstatus), or an activity level, and determining if the status has apreselected relationship with a reference criterion, includes one ormore of analyzing a sample, requesting analysis of the sample,requesting results from analysis of the sample, or receiving the resultsfrom analysis of the sample. (Generally, analysis can include one orboth of performing the underlying method (e.g., an immunoassay) orreceiving data from another who has performed the underlying method.)

Anti-VLA-4 therapy. An anti-VLA-4 therapy is a molecule, e.g., a smallmolecule compound or protein biologic (e.g., an antibody or fragmentthereof, such as an antigen-binding fragment thereof) that blocks VLA-4activity. The molecule that is the anti-VLA-4 therapy is a VLA-4antagonist. A VLA-4 antagonist includes any compound that inhibits aVLA-4 integrin from binding a ligand and/or receptor. An anti-VLA-4therapy can be an antibody (e.g., natalizumab (TYSABRI®)) or fragmentthereof, or a soluble form of a ligand. Soluble forms of the ligandproteins for α4 integrins include soluble VCAM-I or fibronectinpeptides, VCAM-I fusion proteins, or bifunctional VCAM-I/Ig fusionproteins. For example, a soluble form of a VLA-4 ligand or a fragmentthereof may be administered to bind to VLA-4, and in some instances,compete for a VLA-4 binding site on cells, thereby leading to effectssimilar to the administration of antagonists such as anti-VLA-4antibodies. For example, soluble VLA-4 integrin mutants that bind VLA-4ligand but do not elicit integrin-dependent signaling are suitable foruse in the described methods. Such mutants can act as competitiveinhibitors of wild type integrin protein and are considered“antagonists.” Other suitable antagonists are “small molecules.”

“Small molecules” are agents that mimic the action of peptides todisrupt VLA-4/ligand interactions by, for instance, binding VLA-4 andblocking interaction with a VLA-4 ligand (e.g., VCAM-I or fibronectin),or by binding a VLA-4 ligand and preventing the ligand from interactingwith VLA-4. One exemplary small molecule is an oligosaccharide thatmimics the binding domain of a VLA-4 ligand (e.g., fibronectin orVCAM-I) and binds the ligand-binding domain of VLA-4. (See, Devlin etal., Science 249: 400-406 (1990); Scott and Smith, Science 249:386-390(1990); and U.S. Pat. No. 4,833,092 (Geysen), all incorporated herein byreference.)

A “small molecule” may be chemical compound, e.g., an organic compound,or a small peptide, or a larger peptide-containing organic compound ornon-peptidic organic compound. A “small molecule” is not intended toencompass an antibody or antibody fragment. Although the molecularweight of small molecules is generally less than 2000 Daltons, thisfigure is not intended as an absolute upper limit on molecular weight.

Combination Therapy or Alternatives to Anti-VLA-4 Therapy. In someembodiments, the anti-VLA-4 therapy, e.g., natalizumab, is administeredwith a second agent, or an alternative therapy can be administeredinstead of the anti-VLA-4 therapy, such as when a patient is determinedto be at higher risk for PML.

Non-limiting examples of second agents for treating multiple sclerosisin combination with the anti-VLA-4 therapy, or alternative agents foruse instead of the anti-VLA-4 therapy, include: fumaric acid salts, suchas dimethyl fumarate;

Sphingosine 1-phosphate (S1P)-antagonists, such as the SIB-blockingantibody Sphingomab; interferons, such as human interferon beta-la(e.g., AVONEX® or Rebif®)) and interferon β-1b (BETASERON® humaninterferon β substituted at position 17; Berlex/Chiron); glatirameracetate (also termed Copolymer 1, Cop-1; COPAXONE® Teva PharmaceuticalIndustries, Inc.); an antibody or a fragment thereof (such as anantigen-binding fragment thereof), such as an anti-CD20 antibody, e.g.,Rituxan® (rituximab), or an antibody or fragment thereof that competeswith or binds an overlapping epitope with rituximab; mixtoxantrone(NOVANTRONE®, Lederle); a chemotherapeutic agent, such as clabribine(LEUSTATIN®), azathioprine (IMURAN®), cyclophosphamide (CYTOXAN®),cyclosporine-A, methotrexate, 4-aminopyridine, and tizanidine; acorticosteroid, such as methylprednisolone (MEDRONE®, Pfizer), orprednisone; CTLA4 Ig; alemtuzumab (MabCAMPATH®) or daclizumab (anantibody that binds CD25); statins; and TNF antagonists.

Glatiramer acetate is a protein formed from a random chain of aminoacids (glutamic acid, lysine, alanine and tyrosine (hence GLATiramer)).Glatiramer acetate can be synthesized in solution from these amino acidsat a ratio of approximately 5 parts alanine to 3 parts lysine, 1.5 partsglutamic acid and 1 part tyrosine using N-carboxyamino acid anhydrides.

Additional second agents, or agents for use in place of the anti-VLA-4therapy, include antibodies or antagonists of other human cytokines orgrowth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,IL-12 IL-15, IL-16, IL-18, EMAP-11, GM-CSF, FGF, and PDGF. Still otherexemplary second agents include antibodies to cell surface moleculessuch as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45, CD69, CD80,CD86, CD90 or their ligands. For example, daclizubmab is an anti-CD25antibody that may ameliorate multiple sclerosis.

Still other exemplary antibodies include antibodies that provide anactivity of an agent described herein, such as an antibody that engagesan interferon receptor, e.g., an interferon beta receptor. Typically, inimplementations in which the second agent includes an antibody, it bindsto a target protein other than VLA-4 or other than an α4 integrin, or atleast an epitope on VLA-4 other than one recognized by natalizumab.

Still other additional exemplary second agents include: FK506,rapamycin, mycophenolate mofetil, leflunomide, non-steroidalanti-inflammatory drugs (NSAIDs), for example, phosphodiesteraseinhibitors, adenosine agonists, antithrombotic agents, complementinhibitors, adrenergic agents, agents that interfere with signaling byproinflammatory cytokines as described herein, IL-1β converting enzymeinhibitors (e.g., Vx740), anti-P7s, PSGL, TACE inhibitors, T-cellsignaling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathloprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof, as described herein, anti-inflammatory cytokines (e.g. IL-4,IL-10, IL-13 and TGF).

In some embodiments, a second agent may be used to treat one or moresymptoms or side effects of MS. Such agents include, e.g., amantadine,baclofen, papaverine, meclizine, hydroxyzine, sulfamethoxazole,ciprofloxacin, docusate, pemoline, dantrolene, desmopressin,dexamethasone, tolterodine, phenytoin, oxybutynin, bisacodyl,venlafaxine, amitriptyline, methenamine, clonazepam, isoniazid,vardenafil, nitrofurantoin, psyllium hydrophilic mucilloid, alprostadil,gabapentin, nortriptyline, paroxetine, propantheline bromide, modafinil,fluoxetine, phenazopyridine, methylprednisolone, carbamazepine,imipramine, diazepam, sildenafil, bupropion, and sertraline. Many secondagents that are small molecules have a molecular weight between 150 and5000 Daltons.

Examples of TNF antagonists include chimeric, humanized, human or invitro generated antibodies (or antigen-binding fragments thereof) to TNF(e.g., human TNF a), such as D2E7, (human TNFα antibody, U.S. Pat. No.6,258,562; BASF), CDP-571/CDP-870/BAY-10-3356 (humanized anti-TNFαantibody; Celltech/Pharmacia), cA2 (chimeric anti-TNFα antibody;REMICADE™, Centocor); anti-TNF antibody fragments (e.g., CPD870);soluble fragments of the TNF receptors, e.g., p55 or p75 human TNFreceptors or derivatives thereof, e.g., 75 kd TNFR-IgG (75 kD TNFreceptor-IgG fusion protein, ENBREL™; Immunex; see, e.g., Arthritis &Rheumatism 37:S295, 1994; J. Invest. Med. 44:235A, 1996), p55 kdTNFR-IgG(55 kD TNF receptor-IgG fusion protein (LENERCEPT™)); enzymeantagonists, e.g., TNFα converting enzyme (TACE) inhibitors (e.g., analpha-sulfonyl hydroxamic acid derivative, WO 01/55112, andN-hydroxyformamide TACE inhibitor GW 3333, -005, or -022); andTNF-bp/s-TNFR (soluble TNF binding protein; see, e.g., Arthritis &Rheumatism 39:S284, 1996; Amer. J. Physiol. —Heart and CirculatoryPhysiology 268:37-42, 1995).

In one implementation, the anti-VLA-4 therapy and the second agent areprovided as a co-formulation, and the co-formulation is administered tothe subject. It is further possible, e.g., at least 24 hours before orafter administering the co-formulation, to administer separately onedose of the anti-VLA-4 therapy formulation and then one dose of aformulation containing the second agent. In another implementation, theanti-VLA-4 therapy and the second agent are provided as separateformulations, and the step of administering includes sequentiallyadministering the anti-VLA-4 therapy and the second agent. Thesequential administrations can be provided on the same day (e.g., withinone hour of one another or at least 3, 6, or 12 hours apart) or ondifferent days.

The anti-VLA-4 therapy and the second agent each can be administered asa plurality of doses separately in time. The anti-VLA-4 therapy and thesecond agent are typically each administered according to a regimen. Theregimen for one or both may have a regular periodicity. The regimen forthe anti-VLA-4 therapy can have a different periodicity from the regimenfor the second agent, e.g., one can be administered more frequently thanthe other. In one implementation, one of the anti-VLA-4 therapy and thesecond agent is administered once weekly and the other once monthly. Inanother implementation, one of the anti-VLA-4 therapy and the secondagent is administered continuously, e.g., over a period of more than 30minutes but less than 1, 2, 4, or 12 hours, and the other isadministered as a bolus. The anti-VLA-4 therapy and the second agent canbe administered by any appropriate method, e.g., subcutaneously,intramuscularly, or intravenously.

In some embodiments, each of the anti-VLA-4 therapy and the second agentis administered at the same dose as each is prescribed for monotherapy.In other embodiments, the anti-VLA-4 therapy is administered at a dosagethat is equal to or less than an amount required for efficacy ifadministered alone. Likewise, the second agent can be administered at adosage that is equal to or less than an amount required for efficacy ifadministered alone.

Kits. Reagents for performing an anti-JCV antibody assay can be providedin the form of a kit. Except for the patient sample, some or allmaterials required for the assay can be provided in the kit. A kit caninclude for example, a substrate, such as a plate with wells coated withJCV antigen substrate, e.g., HPVLP. The plate can be for example a6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a96-well plate or a 384 well plate. The plates provided in a kit can bepre-coated with JCV VLP antigen, such as at 0.4 μg/mL. In one embodimentthe kit includes materials and reagents for use with high-throughputsystems such as SPR (Solid Phase Receptacle) tips for use withbioMerieux systems.

The kit can also include JCV antigen, e.g., HPVLP lyophilized or insolution, such as for use with the confirmation step of the assay. Inone embodiment, the kit includes a JCV cut-off calibrator, an anti-JCVantibody positive control and a JCV negative control, which are samplesof sera, such as human sera. Solutions containing JCV antigen and seracan include a preservative, such as sodium azide, e.g., 0.05%, 0.1%,1.5%, and 2% sodium azide. In one embodiment, a kit featured in theinvention can include one or more reagents for detecting a complexcontaining anti-JCV antibodies bound to antigen, such as HPVLP. Reagentsfor detecting the complex include, for example, a JCV conjugate, acasein sample, a detectable reagent, such as TMB (tetramethylbenzidine),a wash buffer, and a stop reagent.

The JCV substrate can be, for example, an anti-human antibody, such asan enzyme-conjugated anti-human antibody. In one embodiment, the JCVconjugate is an affinity-purified and peroxidase-conjugated donkeyanti-human antibody. In another embodiment, the casein solution containscasein, a surfactant and a non-azide preservative in buffer (e.g.,phosphate buffered saline (PBS)). In another embodiment, the TMBsubstrate solution includes TMB and hydrogen peroxide in buffer. Inanother embodiment, the kit includes a wash buffer, and the wash buffercan contain, for example, surfactant in PBS with non-azidepreservatives. The stop reagent can be, for example, an acid, such assulfuric acid (e.g., 1 M sulfuric acid).

The solutions provided in the kit can be provided at concentrated levelssuch that dilution is required before use. The HPVLP for use in solutionbinding to anti-JCV antibody in a biological sample, such as in theconfirmation step of the two-step assay, can be provided as aconcentration of 2 mg/mL, 1.5 mg/mL, 1 mg/mL, 0.5 mg/mL, for use at, forexample, 10 μg/mL, 5 μg/mL, 1 μg/mL, 0.8 μg/mL, 0.4 μg/mL, 0.2 μg/mL Thewash buffer, for example, can be provided at 10× concentration. The JCVsubstrate (such as an affinity-purified and peroxidase-conjugated donkeyanti-human antibody) can be provided at, for example, 1 mg/mL, 0.8 mg/mLor 0.6 mg/mL, for dilution by, e.g., 1:40,000, 1:30,000, 1:20,000 or1:20,000 prior to use in an anti-JCV antibody detection assay.

Materials for sealing the reaction mixes, such as sealing tape, can alsobe included in the kit.

Reporting of results. The results of the risk-assessment analysis can bereported, such as to a treatment center, or a healthcare provider, or aninsurance provider. In one embodiment, the results of therisk-assessment are stored in a database.

In one embodiment, informational material is provided for performing andinterpreting the risk assessment. The informational material can provideguidance as to where to report the results of the assessment, such as toa treatment center or healthcare provider or database provider. Theinformational material can be provided in a kit or a packet, and caninclude forms for reporting the results of the assessment, includingeach prong of the assessment (information regarding prior treatment withanti-VLA-4 therapies, prior treatment with immunosuppressants, and JCVstatus), and address and contact information regarding where to sendsuch forms or other related information; or a URL (Uniform ResourceLocator) address for reporting the results in an online database or anonline application (e.g., an “app”). In another embodiment, theinformational material can include guidance regarding whether a patientshould receive treatment with an anti-VLA-4 therapy, depending on thepatient's risk of PML according to the results of the risk assessment.

The kit or packet may also include instructions and items for thecollection or transport of a patient sample to a healthcare provider, orfor receiving a sample from a healthcare provider, or for performing theevaluative methods described herein. For example, besides instructionalinformation, a kit or packet featured in the invention can include oneor more of a swab or scraper, or a vessel (e.g., a cup, a test tube, anampoule, or a bag) for collecting, and storing and transporting abiological sample. The kit or packet may also contain supplies forperforming an immunoassay or a sequencing assay for detection of JCVantibodies or nucleic acids, respectively.

A kit can include one or more containers for the reagents required foran assay, e.g., a JCV-detection assay. The reagents can be provided in aconcentration suitable for use in the assay or with instructions fordilution for use in the assay. In some embodiments, the kit containsseparate containers, dividers or compartments for the assay components,and the informational material. For example, the assay components can becontained in a bottle or vial, and the informational material can becontained in a plastic sleeve or packet. In other embodiments, theseparate elements of the kit are contained within a single, undividedcontainer. For example, an assay reagent is contained in a bottle orvial that has attached thereto the informational material in the form ofa label. In some embodiments, the kit includes a plurality (e.g., apack) of individual containers, each containing one or more unit forms(e.g., for use with one assay) of an assay component. For example, thekit includes a plurality of ampoules, foil packets, or blister packs,each containing a single unit of assay reagent for use in a screening orconfirmatory assay. The containers of the kits can be air tight and/orwaterproof. The container can be labeled for use.

The informational material of a kit or packet is not limited in itsform. In many cases, the informational material, e.g., instructions, isprovided in printed matter, e.g., a printed text, drawing, and/orphotograph, e.g., a label or printed sheet. However, the informationalmaterial can also be provided in other formats, such as computerreadable material, video recording, or audio recording. In anotherembodiment, the informational material of the kit is contactinformation, e.g., a physical address, email address, website, ortelephone number, where a user of the kit or packet can obtainsubstantive information about how to find the information required forthe risk assessment analysis, e.g., where and how to identify priortreatments administered to a subject, and how to perform an assay todetermine the JCV status of a patient. The informational material canalso be provided in any combination of formats.

In some embodiments, a biological sample is provided to an assayprovider, e.g., a service provider (such as a third party facility) or ahealthcare provider, who evaluates the sample in an assay and provides aread out. For example, in one embodiment, an assay provider receives abiological sample from a subject, such as a plasma, blood or serumsample, and evaluates the sample using an assay described herein, anddetermines that the sample contains JCV antibodies or nucleic acid. Insome embodiments, the assay provider, e.g., a service provider orhealthcare provider, can further determine, e.g., by contacting ahealthcare provider or a database service provider, the amount of prioranti-VLA-4 therapy that a patient has received or whether a patient haspreviously received treatment with an immunomodulator. The assayprovider can further determine that the subject is not a candidate toreceive treatment with an anti-VLA-4 therapy, such as natalizumab, orthat the subject is a candidate to receive treatment with animmunomodulator, or that the subject may be a candidate who should haveenhanced monitoring as compared to a subject who is determined to have anegative JCV status (e.g., who tests negative for JCV nucleic acid oranti-JCV antibodies). For example, a candidate who has received priortreatment with an anti-VLA-4 therapy for 24 months or less, and who hasnot received prior therapy with an immunosuppressant, but who isdetermined to be JCV positive, can be selected as a candidate to receivefurther anti-VLA-4 therapy, but with a recommendation to monitor thepatient more frequently for the development of adverse symptoms, such assymptoms that may indicate the development of PML.

In one embodiment, the assay provider performs an assessment for PMLrisk as described herein and determines that subject is a candidate toreceive treatment with an anti-VLA-4 therapy, such as natalizumab. Inone embodiment, the assay provider informs a healthcare provider thatthe subject is a candidate for treatment with the anti-VLA-4 therapy,and the candidate is administered the anti-VLA-4 therapy. For example,the assay provider may determine that a patient is at a lower risk forPML and subsequently inform the healthcare provider of the determinationof the lower risk and that the subject is a candidate for treatment withthe anti-VLA-4 therapy.

In another example, the assay provider determines that a patient is at ahigher risk for PML and subsequently informs a healthcare provider ofthe determination of the higher risk, and recommends that the patient isa candidate for treatment with the anti-VLA-4 therapy, but that thepatient should undergo increased testing for PML and, optionally, JCVstatus. In one embodiment, the assay provider informs the healthcareprovider that the patient is at higher risk of PML and therefore thepatient should receive an alternative to anti-VLA-4 therapy, or thepatient is a candidate to receive anti-VLA-4 therapy with increasedtesting for PML and, optionally, JCV status.

The assay provider can provide the results of the risk assessment, andoptionally, conclusions regarding one or more of diagnosis, prognosis,or appropriate therapy options to, for example, a healthcare provider,or patient, or an insurance company, in any suitable format, such as bymail or electronically, or through an online database. The informationcollected and provided by the assay provider can be stored in adatabase.

In one embodiment, a healthcare provider or insurance provider oranother entity recommends, e.g., to the patient or a second healthcareprovider, that a patient undergo a risk assessment for PML as describedherein.

PML risk stratification tools are useful as one component in makingindividual benefit-risk treatment decisions for patients taking orconsidering taking a VLA4 inhibitor or other therapeutics known toincrease risk of developing PML. Quantification of a patient's PML riskcan be used, for example, in benefit-risk analysis.

Headings, e.g., (a), (b), (i) etc, are presented merely for ease ofreading the specification and claims. The use of headings in thespecification or claims does not require the steps or elements beperformed in alphabetical or numerical order or the order in which theyare presented.

The invention is further illustrated by the following examples, whichshould not be construed as further limiting.

EXAMPLES Example 1. PML Risk in MS Patients was Quantified for the FirstTime Using the Two Established Risk Factors and Anti-JCV Antibody Statusas Determined by a Unique, Two-Step VP1 VLP-Based ELISA

Methods

Patients, Samples and Data Collection

Due to the infrequent occurrence of PML, data on natalizumab-treated PMLpatients were collected from several sources including post-marketingdata from the Biogen Idec global natalizumab safety database andclinical trials as of Mar. 4, 2011. Prior immunosuppressant use datawere not available for all patients exposed to natalizumab; therefore,the proportion of patients with and without prior immunosuppressant usein the TYSABRI® Global Observational Program in Safety (TYGRIS;NCT00477113, NCT00483847) was used as an estimate for the overallnatalizumab-treated population. TYGRIS is an observational cohort studydesigned to obtain long term safety data in natalizumab-treated MSpatients in a clinical practice setting. Assessment of anti-JCV antibodyprevalence in the general MS population was based on a single baselineplasma or serum sample collected from patients from four sources,including ongoing or completed natalizumab clinical studies (AFFIRM(Polman et al., N. Engl. J. Med. 354:899-910, 2006; STRATIFY-1(NCT01070823), TYGRIS-US, and an independent MS registry in Sweden(available on the internet at msreg.net/cms/sv/home, accessed Feb. 3,2011)). A clinical plan was developed for wide-scale collection of serumand plasma samples obtained prior to PML diagnosis, including bothclinical trial and post-marketing cases.

Identification of Natalizumab Treatment Duration as a Risk Factor forPML

Estimates of PML incidence since natalizumab market reintroduction werecalculated based on natalizumab post marketing exposure through Feb. 28,2011, and the number of confirmed PML cases as of May 2011. PMLincidence for each time period (cumulative duration or 12-monthtreatment interval) was calculated using the number of patients thatdeveloped PML during that time period divided by the number of patientsever exposed to natalizumab for that amount of time.

Identification of Prior Immunosuppressant Use as a Risk Factor for PML

Immunosuppressant treatment histories of natalizumab-treated MS patientsthat developed PML in the post-marketing setting and clinical trialswere obtained from the Biogen Idec global natalizumab safety database asof Nov. 2, 2010, and compared to the data obtained from TYGRIS. Thiscut-off date for prior immunosuppressant use history was chosen (versusMar. 4, 2011 for all other data) as it is expected that the inclusion ofthis as a risk factor in labeling (December 2010 in the EU) (PackageInsert. TYSABRI® (natalizumab). Biogen Idec. Weston, Mass. July 2010;Summary of Product Characteristics. TYSABRI® (natalizumab). Biogen Idec.Weston, Mass., Dec. 13, 2010) would be a confounding factor.

Identification of Anti-JCV Antibody Status as a Risk Factor for PML

The overall prevalence of anti-JCV antibodies in the general MSpopulation was determined using a unique two-step VP1 VLP-based ELISA aspreviously described (Gorelik et al., Ann. Neurology, 2010). Theprevalence of anti-JCV antibodies in MS patients with PML where pre-PMLserum or plasma samples were available prior to diagnosis was alsodetermined using this assay and compared to the overall prevalence inthe general MS population.

Estimation of PML Incidence by Anti-JCV Antibody Serostatus

The incidence of PML in anti-JCV antibody positive and negative patientswas estimated using the overall global incidence of PML, and theincidence after 25-48 infusions (the time point after which the increasein PML incidence was most pronounced in this analysis), the anti-JCVantibody prevalence in the general MS population, and the number of MSpatients with PML who had pre-PML samples available that tested anti-JCVantibody positive prior to diagnosis. A one-sided Fisher's exact testwas used to compare the estimated incidence of PML in anti-JCV antibodypositive and anti-JCV antibody negative patients. To provide aconservative estimate of PML incidence in anti-JCV antibody negativepatients, a sensitivity analysis was performed to assess the impact of ahypothetical anti-JCV antibody negative PML case on this estimate.Additionally, a sensitivity analysis was performed to assess thestatistical certainty of this estimate by varying the number of anti-JCVantibody positive PML cases.

Quantification of PML Risk: Prior Immunosuppressant Use, NatalizumabTreatment Duration, and Anti-JCV Antibody Positive Status

Risk factor algorithms were developed to estimate PML incidence inpatients with and without certain risk factors for natalizumabassociated PML and anti-JCV antibody serostatus. These algorithmsestimated PML risk by prior immunosuppressant use (yes or no),natalizumab treatment duration (1-24 months and 25-48 months), andanti-JCV antibody status. These risk algorithms were based upon PMLincidence data by natalizumab treatment duration (1-24 or 25-48 months)and estimates of prior immunosuppressant use in natalizumab-treatedpatients from TYGRIS and in those with PML. In addition, the estimatedoverall prevalence of anti-JCV antibodies in the general MS populationwas used to impute the incidence of PML associated with serostatus forthe three-factor risk algorithm, assuming that all confirmed cases ofnatalizumab-associated PML were anti-JCV antibody positive prior todiagnosis. A sensitivity analysis was performed to quantify the effectof varying the estimates used to develop this three factor algorithmbased upon the highest and lowest values observed.

Results

Identification of Natalizumab Treatment Duration as a Risk Factor forPML

On a worldwide basis, 102 confirmed cases of PML were identified as ofMar. 4, 2011. Overall, PML risk increased with increasing treatmentduration (FIG. 1A), with the greatest increase in risk occurring aftertwo years of therapy, peaking at 1.68 cases per 1000 patients in yearthree (FIG. 1B). Data beyond four year were limited.

Identification of Prior Immunosuppressant Use as a Risk Factor for PML

Prior immunosuppressant use was more common in natalizumab treatedpatients who developed PML compared to patients enrolled in TYGRIS,which represented the overall patient population receiving natalizumab.Forty-five percent of natalizumab-treated PML patients had received oneor more immunosuppressant therapies prior to initiating treatment withnatalizumab, compared with 20.3% in natalizumab-treated patients (13.9%in the US and 23.6% in the EU) from TYGRIS. The most common priorimmunosuppressants used in both the natalizumab-treated PML populationand in TYGRIS included mitoxantrone, methotrexate, cyclophosphamide,azathioprine, and mycophenolate with no specific pattern being observedin the type of immunosuppressant, duration of use, or wash-out periodbetween discontinuation of the immunosuppressant and initiation ofnatalizumab (Table 1).

TABLE 1 Prior immunosuppressant use history in natalizumab-associatedPML patients and patients enrolled in TYGRIS. All post-marketingconfirmed PML TYGRIS cases with prior patients with priorimmunosuppressant use immunosuppressant use Characteristics (N = 32) (N= 792) Prior immunosuppressants  Mitoxantrone 18 (56%) 344 (43%) Methotrexate 5 (16%) 45 (6%)  Azathioprine 5 (16%) 133 (17%) Cyclophosphamide 6 (19%) 71 (9%)  Mycophenolate 4 (13%) 48 (6%)  Other3 (9%) 201 (25%) Duration of Prior immunosuppressant use  Range 0.03-204months <1-24 months  Mean 30.6 months 10.1 months Wash-out period  Range2-93 months <1-24 months  Mean 24.7 months 8.5 months

Quantification of PML Risk: Natalizumab Treatment Duration and PriorImmunosuppressant Use

When patients were stratified by natalizumab treatment duration (1-24 or25-48 months) and prior immunosuppressant use (yes or no), four distinctsubgroups of patients were identified with respect to incidence of PML(FIG. 2). Three of these subgroups had an estimated PML incidence ofless than or approximately equal to 1 per 1000. PML risk was lowest inpatients who were treated with natalizumab for 1-24 months, and who hadnot received prior immunosuppressant therapy, 0.19 per 1000 (95% CI:0.10-0.33). The fourth subgroup, including patients who had both ofthese risk factors for PML, had the highest risk, with an estimated PMLincidence of 4.3 per 1000 (95% CI: 2.9-6-2).

Identification of Anti-JCV Antibody Status as a Risk Factor for PML

5,896 patients from AFFIRM, TYGRIS-US, STRATIFY-1, and the Swedish MSRegistry had a baseline sample available for anti-JCV antibody testing.Demographics, including natalizumab treatment duration and priorimmunosuppressant use history, where available, were similar betweenthese data sources (Table 2). The overall anti-JCV antibody prevalencein the general MS population assessed in this study was 55% (95% CI:54-56%).

TABLE 2 Anti-JCV antibody prevalence and demographic data of general MSpopulation. AFFIRM TYGRIS-US STRATIFY-1 Swedish MS (N = 823) (N = 1480)(N = 1096) Patients (N = 2497) Anti-JCV Antibody 54.6% 47.6% 56.0% 59.0%Prevalence (51.1-58.0) (45.0-50.1) (53.0-59.0) (57.0-60.9) Age (years) Range 18-50 18-75 12-75 12-75  Mean 35.9 44.3 44.4 37.5  Median 36 4445 37 Gender (%)  Male 30.6% 24.1% 24.3% 28.1%  Female 69.4% 75.9% 75.7%71.9% Geography North US US Sweden America and and EU/Rest of CanadaWorld Prior immunosuppressant Use (%)  Yes 3.6% 8.8% 3.8% NA  No 96.4%91.2% 96.2% NA

In the TYGRIS-US dataset, 1451 of the 1480 patients had age, gender andprior immunosuppressant information available. In the STRATIFY-1dataset, 988 of 1096 patients had prior immunosuppressant informationavailable. In the Swedish MS dataset, 2464 of 2497 patients had ageinformation available and 2494 of 2497 patients had gender informationavailable. Prior immunosuppressant use was not available (NA) in theSwedish MS patients.

One or more pre-PML samples were obtained from 25 natalizumab-treated MSpatients 6.5-187 months prior to PML diagnosis. As shown in SupplementalTable 1, these 25 patients had clinical characteristics that weresimilar to the 102 confirmed PML cases worldwide, indicating no obviousselection bias. All patients for whom multiple pre-PML samples wereavailable tested anti-JCV antibody positive at all time points,including those samples collected prior to initiation of natalizumabtreatment. The 100% (25 out of 25) anti-JCV antibody positive prevalencein MS natalizumab-treated PML patients prior to PML diagnosis wassignificantly different from the expected 55% prevalence observed in thegeneral MS population (p<0.0001), demonstrating the ability of anti-JCVantibody status to serve as an additional PML risk stratification tool.

Supplemental Table 1 Clinical characteristics of 25 MS PML patients withPre-PML samples compared to all 102 post-marketing PML cases. MSpatients with pre-PML All post-marketing samples - all tested positiveconfirmed PML cases Characteristics for anti-JCV antibody (N = 25) (N =102) Geographic distribution  US 4 (16%) 42 (41%)  Europe/ROW 21 (84%)60 (59%) Age at diagnosis  Range 27-55 23-67  Mean 40.7 44.6  Median 4144 Gender  Male 8 (32%) 32 (31%)  Female 17 (68%) 70 (69%) Duration ofMS at diagnosis (years)  Range 1.5-21 1.5-23  Mean 12.2 11.9  Median12.3 11.1 TYSΛBRI ® exposure (month)  Range 17-51 12-52  Mean 33.0 30.8 Median 32 30 Prior immunosuppressants  Yes 9 (38%) 39 (42%)  No 15(63%) 54 (58%)

MS onset date was unknown for 5 patients in the anti-JCV antibodypositive group and 38 patients in all PML group. Prior immunosuppressantuse status was unknown for 1 patient in anti-JCV antibody positive groupand 9 patients in the overall PML group.

Estimation of PML Incidence by Anti-JCV Antibody Serostatus

The incidence of PML in patients who were anti-JCV antibody positive wasestimated to be almost 2-fold that of the overall natalizumab-treatedpopulation (Table 4). To estimate the overall incidence of PML byanti-JCV antibody status, the following method was used: Based on the 25natalizumab-treated MS patients with pre-PML samples available, it wasestimated that the 25 MS patients with PML came from approximately 20276patients receiving natalizumab treatment, based upon the overall rate ofPML, 1.23 per 1000 patients (FIG. 1). Assuming that 55% of these 20276patients were anti-JCV antibody positive (i.e., 11152 patients) and 45%were anti-JCV antibody negative (i.e., 9124 patients), the incidence ofPML in anti-JCV antibody positive patients was estimated to be 2.24cases per 1000 patients treated (=1000×25/11152), 95% CI: 1.45-3.31,similar to the rate estimated in the literature (Tyler, Ann. Neurol.68:271-274, 2010). Conversely, the estimated incidence of PML inanti-JCV antibody negative patients was 0 cases per 1000 patients (95%CI: 0-0.40), significantly different from the estimated incidence inanti-JCV antibody positive patients, p<0.0001.

TABLE 4 Estimated incidence of PML by anti-JCV antibody status based on25 cases of PML that were anti-JCV antibody positive prior to the onsetof PML. Number of Total Patients PML Cases Treated Incidence per 1000patients (95% CI) Overall PML incidence in natalizumab-treated MSpatients Anti-JCV Antibody 25 11152 2.24 (1.45, 3.31) Positive Anti-JCVAntibody 0 9124 0 (0, 0.40) Negative Total 25 20276 1.23 (0.80, 1.82)P-value <0.0001 RR (95% CI) ∞ (6.44, ∞) PML incidence after 25-48 monthsof natalizumab therapy Anti-JCV Antibody 18 4533 3.97 (2.36, 6.27)Positive Anti-JCV Antibody 0 3709 0 (0, 0.99) Negative Total 18 82422.18 (1.30, 3.45) P-value <0.0001 RR (95% CI) ∞ (5.63, ∞) Sensitivityanalysis: assumption of 1 anti-JCV antibody negative hypothetical PMLpatient Anti-JCV Antibody 25 11598 2.16 (1.40, 3.18) Positive Anti-JCVAntibody 1 9489 0.11 (0.00, 0.59) Negative Total 26 21087 1.23 (0.81,1.81) P-value <0.0001 RR (95% CI) 20.5 (3.35, 842)

The effect of natalizumab treatment duration on this estimate wasassessed in an analysis using the PML incidence after 25-48 months ofnatalizumab (the duration of therapy after which the increase inincidence was most pronounced). The risk of PML after 25-48 months oftherapy in anti-JCV antibody positive patients was 3.97 per 1000 (95%CI: 2.36-627) and in anti-JCV antibody negative patients was 0 per 1,000(95% CI: 0-0.99), Table 3.

At the time of this writing, the incidence of PML in anti-JCV antibodynegative patients could not be fully ascertained because no PML case hastested anti-JCV antibody negative prior to diagnosis. Therefore, toestimate the incidence of PML in anti-JCV antibody negative patients, asensitivity analysis was performed assuming a hypothetical case of PMLhad occurred in an anti-JCV antibody negative patient, thus allowing fordetermination of a conservative estimate for which the rate is likelylower. This analysis demonstrated an estimated incidence of PML inanti-JCV antibody negative patients of at least 20-fold lower than inanti-JCV antibody positive patients, p<0-0001 (Table 3).

Sensitivity analysis of the effect of increasing the number of anti-JCVantibody positive PML patients with Pre-PML samples available thattested anti-JCV antibody positive demonstrated that statisticalcertainty regarding the increased risk of PML in anti-JCV antibodypositive patients was not improved beyond 25 available Pre-PML samples(Table 5).

TABLE 5 Effect of increasing anti-JCV antibody positive PML case numberson statistical certainty of PML incidence estimations in anti-JCVantibody positive patients. Number of anti-JCV antibody positive PML PMLIncidence per 1000 Patients cases that tested prior to Anti-JCV antibodynegative Anti-JCV antibody positive PML diagnosis Incidence 95% CIIncidence 95% CI 1-sided p-value: 10 0, 1.01 1.08, 4.12 p = 0.025 25 00, 0.40 2.24 1.45, 3.31 P <0.0001 30 0, 0.34 1.51, 3.20 40 0, 0.25 1.60,3.05 50 0, 0.20 1.66, 2.95 60 0, 0.17 1.71, 2.88

Quantification of PML Risk: Natalizumab Treatment Duration, PriorImmunosuppressant Use, and Anti-JCV Antibody Status

A combined, quantitative PML risk algorithm was developed fornatalizumab-treated MS patients based on natalizumab treatment duration,prior immunosuppressant use, and anti-JCV antibody status (FIG. 3).Because JCV exposure is a requirement for PML, patients who are anti-JCVantibody negative represented the lowest risk sub-group in the PML riskstratification algorithm, with an estimated risk of <0.11 per 1000 (95%CI: 0-0.59), based on the conservative estimate determined in thesensitivity analysis. Conversely, the highest risk group consists ofthose patients that are anti-JCV antibody positive, with priorimmunosuppressant use, and who have been treated with natalizumab for25-28 months. This algorithm assumed that all 102 confirmed cases of PMLwere anti-JCV antibody positive prior to PML diagnosis. For the higherrisk sub-group (patients who had all three risk factors) the estimatedPML risk was approximately 7.8 per 1000 (95% CI: 5·2-11·3). For patientswho were anti-JCV antibody positive with no prior immunosuppressant use,PML risk was consistent with risk in the overall natalizumab-treatedpopulation at similar time points (FIGS. 1A and 1B). Sensitivityanalysis of the effect of varying the estimates used to develop thisalgorithm resulted in minimum and maximum values that were generallyconsistent with the original estimates seen in the base case scenario(see FIGS. 4A and 4B). The risk in anti-JCV antibody negative patientswas determined by assuming a hypothetical case of PML had occurred in ananti-JCV antibody negative patient.

This analysis varied anti-JCV antibody prevalence in the general MSpopulation from 48% (as seen in TYGRIS-US) to 59% (as seen in theindependent Swedish Registry), prior immunosuppressant use in thenatalizumab-treated MS population from 14-24% (based upon US and EUestimates in TYGRIS, respectively), and natalizumab 25-48 month exposureestimates from 35-45% (based upon the current estimate of 40% and anapproximate 10% increase over the past year). Plots represent pointestimates and 95% confidence intervals for each scenario (base case,minimum, and maximum). In general, the base case scenario was relativelyconsistent with the minimum and maximum estimates.

Since JCV infection is required for PML development, the lowest risk ofPML was determined to be in patients who were anti-JCV antibodynegative, <011 cases per 1000 natalizumab-treated patients (95% CI0-0.59), irrespective of other risk factors, at least 20-fold lower thanin anti-JCV antibody positive patients, p<0.0001. Although there havebeen no cases of PML in anti-JCV antibody negative patients to date, thetrue risk of developing PML cannot be zero because the anti-JCV antibodyassay has an estimated analytical false-negative rate between2.5-3.2%.^(1,19) Conversely, PML risk was highest in patients whopossessed all three risk factors (natalizumab treatment for 25-48months, prior immunosuppressant use, and anti-JCV antibody positivestatus) with an estimated incidence of 7.8 cases per 1000 patients (95%CI: 5·2-11·3).

Example 2. The Anti-JCV Antibody ELISA was Validated at ClinicalLaboratories to Demonstrate the Robustness of the Method

A novel, 2-step enzyme-linked immunosorbent assay (ELISA) that detectsanti-JCV antibodies in human serum or plasma was recently described (seePCT/US2011/020832). The key attributes of the assay include both directbinding and in-solution competition components; use of wellcharacterized preparations of JC virus-like particles (VLP); inclusionof appropriate quality control (QC) samples; statistical determinationof assay cut points using a large number of longitudinally collectedclinical samples; normalization of the signal of the assay; anddetection of several isotypes of anti-JCV antibodies (including IgG,IgM, IgA, and IgE).

The anti-JCV antibody ELISA was validated at three clinical laboratoriesin order to demonstrate the robustness of the method. Analyticalvalidation was performed by evaluation of intra- and inter-assayprecision, analytical specificity and sensitivity, matrix interference,robustness, and reagent stability.

Stability of anti-JCV antibodies in serum and plasma samples wasdemonstrated by using assay QC samples prepared from pooled human seraas well as serum and plasma samples from individual donors. Anti-JCVantibodies were shown to be stable in serum or plasma through 6freeze/thaw cycles, and for 14 days when stored both at ambient (18-25°C.) temperature and at 2-8° C. Additionally, stability of anti-JCVantibodies in whole blood stored at 2-8° C., at ambient temperatures(18-25° C.), or at 37° C. for 7 days, 7 days, and 3 days, respectively,prior to processing was also shown using both serum and plasmacollection tubes. Stability of the JC VLP was shown through 4freeze/thaw cycles and for 18 months at 2-8° C.

The analytical validation demonstrated that the assay is sensitive,specific, and precise. The assay sensitivity was estimated at 1.7 ng/mLusing a humanized anti-JCV monoclonal antibody control, and wasestimated at 1.25 μg/mL using a purified polyclonal antibody fromanti-JCV antibody positive sera. The sensitivity to detect JCV infectionwas estimated to be 97.5%. The specificity of the assay to discriminateJCV specific antibodies from antibodies directed to BK virus, a relatedpolyomavirus, was also demonstrated. The average inter- and intra-assayprecision was approximately 6.4% and 12.2% for the screening step and2.6% and 5.3% for the confirmation step. Results obtained for plasma andserum were highly congruent, and assay robustness was demonstrated bythe highly concordant results generated by 3 laboratories testing apanel of 100 blinded samples.

Example 3. A Refined Two-Step JCV Assay (the Gen2 Assay) Provides MoreAccurate Results than the Original Assay (Gen1Assay)

The two-step anti-JCV antibody assay was modified following optimizationrounds. The new assay differs from the first in at least the followways:

-   -   HPVLP is used at a substrate concentration of 0.4 μg/mL on        plates in the first step, and in solution in the confirmatory        assay, as opposed to 1 μg/mL used in the Gen1 assay;    -   Patient serum is diluted 1:101 prior to applying to HPVLP on        plates in the first step of the assay, or to HPVLP in solution        in the confirmatory assay, as opposed to 1:200 in the Gen1        assay;    -   The secondary reagent (anti-human IgG) conjugated to HRP is        typically diluted 1:20,000 (but may have to be readjusted for        new lots to match signal to previous lot), and the incubation        time with the conjugate is only 30 min. In the Gen1 assay, the        same reagent was diluted 1:80,000 and incubation time was 60        min;    -   the binding reaction is assayed by incubating the HRP substrate        TMB for 20 minutes±2 minutes, whereas in Gen1, the TMB        incubation was for 20 minutes±5 minutes;    -   In the confirmation assay, 10 μl of sample is added to 1 mL of        confirmation buffer (1:101 dilution), and the reaction proceeds        for 10 to 20 minutes. In the Gen1 assay, a 2× concentration of        sample (1:100 dilution) and HPVLP (2 μg/mL) was mixed in equal        proportion and then incubated for 60 minutes.    -   The cut-off calibrator (CO) is adjusted to have a reactivity        index of about nOD 1.0, and a positive control (PC) is adjusted        to have a reactivity index of about nOD 1.3). The CO and PC are        made by mixing an anti-JCV antibody positive serum and an        anti-JCV antibody negative serum. For the negative control (NC),        which is typically bottle negative sera, the reactivity index        target is about 0.1. Qualitatively, the controls come from        different pools of human serum, but from an assay target        concentration, they are similar to the Gen 1 control levels.

The JCV Gen2 clinical agreement study results are summarized in Table 6below. All Gen1 testing was performed at a Focus Diagnostics referencelaboratory (Cypress, Calif.), and the testing sites for the Gen2 assayincluded Denver (n: overall=275; on TYSABRI®=149; Naïve=126); New York(n: overall=275; on TYSABRI®=136; Naïve=139); and Focus Diagnostics(n=overall 262; on TYSABRI®=95; Naïve=167). Percent agreement isexpressed as (Gen2/Gen1), lower and upper bound of the 96% confidenceinterval (95% Cl: LB to UB).

TABLE 6 JCV Gen2 Clinical Agreement Study Results Overall Patients onTYSABRI ® Naïve Patients Negative % Positive % Negative % Positive %Negative % Positive % Agreement Agreement Agreement Agreement AgreementAgreement Testing Site (NPA) (PPA) (NPA) (PPA) (NPA) (PPA) Denver 86.5%97.2% 85.7% 94.9% 87.3% 100% (115/133) (138/142) (60/70) (75/79) (55/63)(63/63) 95% 95% 95% 95% CI: 87.7 95% 95% CI: 79.6 to CI: 93 to CI: 75.7to to 98% CI: 76.9 to CI: 94.3 to 91.3% 98.9% 92.1% 93.4% 100% New York88.8% 100% 89.5% 100% 88.3% 100% (119/134) (141/141) (51/57) (79/79)(68/77) (62/62) 95% 95% 95% 95% CI: 95.4 95% 95% CI: 82.4 to CI: 97.3 toCI: 78.9 to to 100% CI: 79.3 to CI: 94.2 to 93.1% 100% 95.1% 93.7% 100%Focus 90.2% 100% 87.2% 100% 91.8% 100% Diagnostics (101/112) (150/150)(34/39) (56/56) (67/73) (94/94) 95% 95% 95% 95% CI: 93.6 95% 95% CI:83.3 to CI: 97.5 to CI: 73.3 to to 100% CI: 83.2 to CI: 96.1 to 94.4%100% 94.4% 96.2% 100%

Example 4. Anti-JCV Antibody Status can be Used to Categorize aPatient's Risk for PML

We hypothesized that anti-JCV antibody positive patients could befurther stratified for the risk of developing PML based on anti-JCVantibody titers (nOD or index) and anti-JCV antibody avidity/affinity (%inhibition). This hypothesis was derived from the observation thatpatients having an anti-JCV antibody titer and % inhibition below apredetermined level (“a clinical cut-point”) are at lower risk fordeveloping PML compared to the overall anti-JCV antibody positivepopulation. To determine the relative risk of PML suggested by thestatus of antibody titer and percent inhibition, anti-JCV antibodytiters and percent inhibition could be made prior to initiation ofTYSABRI® (natalizumab) or when patients are already on TYSABRI®.

To determine the relative risk of PML suggested by the status ofantibody titer and percent inhibition in combination with other riskfactors, pre-existing data from two different anti-JCV antibody assays(“Generation I” and “Generation II”) was collected and analyzed. Thepre-existing data included anti-JCV antibody titer information expressedas “nOD” or “index.”

In the Generation I assay, 22% ( 77/356) of anti-JCV antibody positivepatients had nODs>1.0 (C-1801), and 34% ( 13/38) of anti-JCV antibodypositive PML patients had nODs>1.0 (C-1801). Thus ˜1.5 fold more PMLpatients have an nOD>1.0 as compared to non-PML patients. Thistranslates to a 2-3-fold risk ratio associated with an nOD>1.0.

6% of TYSABRI® non-PML patients were also observed to have >2 foldchange in anti-JCV antibody titer (nOD) for longitudinal samplescollected over >2 years (C-1801). However, the majority of PML patientswith longitudinal samples collected at informative timepoints (>1 yearbefore PML diagnosis, and within 6 month of PML diagnosis and at PMLdiagnosis) demonstrated a >2 fold increase in anti-JCV antibody titer(nOD). This suggests that patients who do not exhibit a significantchange in JCV titer over time are at a lower risk of developing PML.

Exemplary nOD and antibody titers are provided in FIGS. 5A-10B. Patientdata is summarized in Table 7.

A graph of the statistical analysis is shown in FIG. 11.

Statistical analysis indicated that for percent inhibition, ˜17% ofantibody-positive samples are less than 0.502, and ˜0% of PML samplesare less than 0.502. ˜30% of antibody-positive samples are less than 70%inhibition, and ˜0% of PML samples (with index <3.0) are less than 70%inhibition.

TABLE 7 anti-JCV antibody titers and nOD measurements in patients.Patient Diagnosis nODs or Index Titers 1 9 Oct. 2009 Ratio: Ratio:1.077/0.258 = ~4 5400/600 = 9 fold between first and fold between firstand second test second test 2 16 Feb. 2005 Consistently high ( >1.0)Consistently high (16200) 3 8 Oct. 2009 Ratio: 0.385/0.129 = ~3 foldRatio: 600/200 = 3 between first and second test between first andsecond test (increased) (increased) 4 16 Feb. 2010 Ratio: 0.628/0.309 =~2 fold Ratio: 1800/600 = 3 between first and second test between firstand second test (increased) (increased) 5 14 Jun. 2009 No increase Noincrease 6 Unknown No increase No increase 7 11 Jul. 2008 Ratio:0.639/0.226 = ~2.8 Ratio: 1800/200 = 9 between first and second testbetween first and second test (increased) (increased)

Example 5. An Analytically Validated Anti-JCV-Antibody Assay has beenIntroduced into Clinical Practice to Stratify MS Patients for Higher orLower Risk of PML

The aim of the below study was to assess anti-JCV-antibody titer changesprior to and after initiation of treatment with natalizumab.

The anti-JCV antibody assay (Gorelik et al., Ann. Neurol. 2010) wasapplied to samples of Swedish MS patients treated with natalizumab,including five PML positive patients. Normalized OD (nOD) values of theanti-JCV antibody assay were studied before and during treatment withnatalizumab. Positive samples were diluted in 1:3 dilution steps todetermine titer levels. A proportion of the same patients was alsotested for antibodies towards a nuclear human cytomegalovirus (CMV)antigen (Schmitz et al., J. Clin. Microbiol. 1977), and antibodiestowards the recombinant varicella-zoster (VZV) glycoprotein E antigen(Thomsson, J. Virol. Methods 2011).

TABLE 8 Patients tested for anti-JCV antibodies. Characteristics FemaleMale All Patients (n, %) 603 (70%) 258 (30%) 861 Age (median, range) 37(13-60) 36 (12-63) 36 (12-63) Time between 12 (1-38) 12 (1-36) 12 (1-38)paired sampling (median, range) Patients with time between paired 244(28%) sampling 1 to 8 month (n, % all patients) Patients with timebetween paired 296 (34%) sampling 8 to 18 month (n, % all patients)Patients with time between paired 321 (37%) sampling >18 month (n, % allpatients)

After initiation of natalizumab treatment, the anti-JCV antibody levelsremained relatively stable with a mild decline of nOD levels observed inthe anti-JCV positive patients. The apparent decline in anti-JCVantibody levels (nOD) was observed when patients were on natalizumabtreatment (n=471), but not during the preceding interferon beta therapy(n=210). This indicates a potential effect of natalizumab therapy on theanti-JCV antibody levels, without significantly affecting theserological status and the seropositivity rate (pre: 56%; post 55%).

After initiation of natalizumab treatment, the anti-VZV (OD) (n=715),but not the anti-CMV (n=502) antibody levels declined slightly.

For the 5 patients who developed PML, the observed change in levels ofanti-JCV antibodies (nOD) in serum is summarized in the below Table 9.Levels of anti-JCV antibodies (nOD) in serum increased at the time ofPML diagnosis compared with the baseline values.

TABLE 9 Change in nOD during natalizumab treatment Months treated withΔnOD between time of initiation of PML natalizumab prior to PMLnatalizumab treatment and time of Patient diagnosis PML diagnosis 1 ~250.348 2 ~29 0.284 3 ~34 0.190 4 ~49 0.932 5 ~25 0.175

From this study we concluded that the therapy with natalizumab may leadto a mild decrease in anti-JCV antibody levels (nOD) without affectingthe JCV seropositivity rate. Notably, only 5% of the anti-JCV positivereference population demonstrated a change in nOD values (ΔnOD) above0.151 (95%-percentile), while this was observed in all the 5 Swedishcases of PML at time of diagnosis compared with the baseline values.Thus, the investigation of an increase in anti-JCV antibody levelsduring natalizumab therapy, prior to PML diagnosis, in the context ofPML risk stratification is warranted.

Example 6. Use of a Clinical Cut-Off Distinct from an Analytical Cut-Offto Delineate High and Low Risk Groups Among Anti-JCV Antibody PositivePatients

Results from the Stratify I study were used to determine a clinicalcut-off distinct from an analytical cut-off to delineate high and lowrisk groups among anti-JCV antibody positive patients. Thus, a patient'srisk of PML would be initially based on baseline anti-JCV antibody titerlevels. The Generation II anti-JCV antibody assay was used in thisstudy.

TYSABRI® non-PML patients (Stratify I, n=1044) and PML patients (>6months prior to PML diagnosis (n=38) were evaluated (FIG. 12). In theGeneration II assay, 17% of anti-JCV antibody positive patients hadtiters (index) below the lowest titer (index) observed for samples fromPML patients collected >6 months prior to PML diagnosis, suggesting thatthose patients may have lower risk for developing PML (like anti-JCVantibody negative patients). Additionally 50% of anti-JCV antibodypositive patients had titers (index) below index 1.5, compared to only13% PML patients from whom samples were collected >6 months prior to PMLdiagnosis had index <1.5. Also, only 4.4% of the PML patients not knownto previously receive immunosuppressant agents had samples with index<1.5, suggesting that those patients may have lower risk for developingPML compared to patient5s with high anti-JCV antibody titer (nOD orindex).

Patients having an nOD <0.5 (109/1044 (10.4% of total samples) or109/549 (20% of anti-JCV antibody positive patients)) were determined tobe in the lowest PML risk group (potentially as low as anti-JCV antibodynegative patients), as no PML patients had an index<0.5. Patients havingindex>0.5 but <1.5 were determined to be in the lower risk zone, as 50%of non-PML anti-JCV antibody positive patients and only 13% of PMLpatients, respectively, had samples in this zone. Additionally, only 4%of PML patients who were not known to receive prior immunosuppressivetherapies, had samples with index <1.5 (FIG. 13). Patients having anindex >1.5 (271/549 (50%) of anti-JCV antibody positive population) weredetermined to be at higher risk for PML. Forty seven percent of patientswere anti-JCV antibody negative.

Post-PML diagnosis, patients are subjected to immune-adsorption (IA) orplasma exchange (PLEX) to remove circulating natalizumab and to restoreimmune function. The anti-JCV antibody levels are rapidly restored topre-procedure levels in these patients.

Example 7. Proposed Statistical Methodology for Assigning StratifiedRisks to Multiple Sclerosis (MS) Patients Undergoing TYSABRI® TreatmentWho have Already Tested Positive for Anti-JCV Antibodies in the RefinedTwo-Step Anti-JCV Assay Described Herein

For the STRATIFY-II (American Academy of Neurology (AAN) Meeting, Apr.21-28, 2012, abstract 5041.002) study, two alternative strategies(denoted as Strategies 1 and 2) for assigning PML risk to anti-JCVsero-positives will be evaluated. Strategy-1, the more conservative ofthe two methods, will be a refinement of one of the nonparametricbivariate tolerance regions provided in the attached report. Strategy-2,whose statistical methodology is sketched below, should assign a higherproportion of future anti-JCV sero-positives to low risk of developingPML compared with Strategy-1.

Strategy-2 devises a lower simultaneous tolerance region around a fittedequation that measures %-inhibition vs. index for a PML patient sample.Whereas Strategy-1 constructs a low risk region based on the%-inhibition/index measurements from the two-step anti-JCV assay inSTRATA (Ann. Neurol., 68:295-303, 2010) and STRATIFY-I (Ann. Neurol.,70:742-750, 2011) patients (almost all of which are assumed to have verylow risk of developing PML), Strategy-2 constructs a high risk regionbased on measurements collected from MS patients prior to their dates ofPML diagnosis. Though our limited collection of PML samples may not berepresentative of the entire universe of Tysabri treated MS patientsinfected with PML, Strategy-2 assumes that the %-inhibition vs. indexrelationship in these samples is representative of the PML universeprior to diagnosis. This assumption was statistically supported byanti-JCV PML data showing a %-inhibition vs. index relationship parallelto that of the STRATIFY-1+STRATA. It is this parallelism that exploitedby Strategy-2 to model the PML %-inhibition vs. index relationship.

The relationship between %-inhibition and index will be firststatistically modeled for the combined set of STRATIFY-1/STRATA/PMLsamples. The fitted equation to %-inhibition vs. index will distinguishbetween PML and STRATIFY-1/STRATA samples. A lower simultaneous 95% or99% tolerance region will then be constructed around the fitted equationconstrained for PML samples. Future anti-JCV sero-positives with%-inhibition/index measurements falling inside this tolerance regionwill be assigned higher risk of developing PML; this tolerance regionshould guarantee that at least 95% (or 99%) of samples from PML patientsprior to their diagnosis will be assigned to higher risk. Note thatfuture samples with index measurements >2.5 will automatically beassigned to higher risk of developing PML.

Statistical Details. The following mixed model (or some refinement ofit) will be first fit using the SAS MIXED procedure to a combined set ofGen-2 anti-JCV

STRATIFY-1+STRATA+PML sample %-inhibition/index measurements.

Y _(ij)=β₀ +δ×W+β ₁ ×X ⁺+β₁₁ ×X ⁺ ×X ⁺+π_(i)+ε_(ij)   (1)

where

-   -   Y (or log-ratio)=−log_(e) {1−% inhibition/100};    -   X⁺=index if index<x₀        -   =x₀ for x₀≤index≤2.5;    -   W=0 if the sample was collected from a STRATIFY-1 or STRATA        patient        -   =1 if the sample was collected from a PML patient prior to            diagnosis;    -   π_(i)=random effect for patient i;    -   ε_(ij)=random assay measurement+longitudinal error for jth time        point of ith patient;

ε_(ij)'s are assumed normally and independently distributed with meanzero and variance σ²; π_(i)'s are assumed normally and independentlydistributed with mean zero and variance σ_(p) ²; ε_(ij)'s and π_(i)'sare assumed independent. Thus for mixed model in equation (1), X⁺ and Ware fixed effects, while π_(i) and ε_(ij) are random effects. Apreliminary estimate of x₀ was 1.77, but that will be refined. A lower95 or 99% simultaneous tolerance region around the fitted equation willbe constructed (refs. 1-5). Lower boundaries of the tolerance region asa function of index level will then be back transformed into%-inhibition. Future samples whose %-inhibition/index measurementseither fall into the tolerance region or have index measurements >2.5will be judged at higher risk of developing PML.

The below Table 10 provides estimated percentages of anti-JCV antibodypositives that will be classified as lower risk based on different nODs.

TABLE 10 Estimated percentages of anti-JCV antibody positives that willbe classified as lower risk based on different nODs. EstimatedProportion of Pre-PML Estimated Estimated Sero-positives PercentagePercentage misclassified of Future Empirical of Future at Lower PMLpre-PML Empirical Percentage of non-PML Risk (based on Sero- Percentageof non-PML Sero- a fitted positives In-house Index STRATIFY-1 positivesWeibull to 39 Misclassified collection of Measurement Sero-positivesAssigned to independent at Lower pre-PML Rule for Assigned to Lower PMLpatient PML Risk Sero-positives Assignment Lower Risk of Risk withsamples (with 95% misclassified to Lower Developing 95% collected priorConfidence at lower PML PML Risk PML Confidence to diagnosis)**certainty) risk * ≤0.40 11.1% (66/595) ≥9.2% 0.4% ≤1.2% 0% (0/153) ≤0.5016.0% (95/595) ≥13.7% 0.8% ≤2.0% 0.65% (1/153) ≤0.65 22.7% (135/595)≥20.0% 1.7% ≤3.6% 4.58% (7/153) ≤0.70 25.2% (205/595) ≥22.5% 2.0% ≤4.2%4.58% (7/153) ≤0.75 25.7% (153/595) ≥22.9% 2.4% ≤5.0% 8.50% (13/153)≤1.00 34.5% (205/595) ≥31.4% 5.4% ≤9.6% 11.76% (18/153) ≤1.25 41.0%(244/595) ≥37.8% 9.7% ≤15.7% 15.69% (24/153) ≤1.50 46.9% (279/595)≥43.6% 15.6% ≤22.2% 22.22% (34/153) *Biased estimates of pre-PMLpopulation due to multiple and unequal numbers of measurements withinpatient donors. ** Estimated fit based on average of 1000 simulationswhere 1 time point per pre-PML patient was randomly selected.

Other embodiments are in the claims.

1.-85. (canceled)
 86. A method of treating a Multiple Sclerosis (MS)patient, the method comprising acquiring the result of an assay fordetecting JC Virus (JCV) antibodies in a biological sample from thepatient, and responsive to a determination that the sample is negativefor anti-JCV antibodies, administering an anti-VLA-4 therapy, whereinthe assay comprises: a) forming a first reaction mixture comprising afirst aliquot of said sample and a substrate on which is disposed highlypurified viral-like particles (HPVLP); b) detecting the level ofanti-JCV antibody bound to said substrate on which is disposed HPVLP bydetecting a labeled detection reagent bound to anti-JCV antibody boundto said substrate, evaluating a cut-off calibrator having a score ofabout 1, a positive control having a score of about 1.3, and a negativecontrol having a score of about 0.1, and assigning to the sample a valueindicative of the level of anti-JCV antibody; c) responsive to a levelof anti-JCV antibody corresponding to a nOD value between 0.2 and 0.4 instep b), forming a second reaction mixture containing a second aliquotof said sample and solution-phase HPVLP, and detecting the level ofunbound antibody in said second reaction mixture, by detecting anti-JCVantibody capable of binding with a substrate on which is disposed HPVLP;d) forming a third reaction mixture containing a third aliquot underconditions where anti-JCV antibodies in the sample are not bound byHPVLP or other antigen, and detecting the level of unbound anti-JCVantibody in the third reaction mixture by detecting anti-JCV antibodycapable of binding with a substrate on which is disposed HPVLP; and e)determining the level to which the presence of HPVLP in the secondreaction mixture inhibits the level of unbound anti-JCV antibody in saidsecond reaction as compared with the level of unbound antibody in saidthird reaction mixture, wherein said sample is classified as negativewhen said inhibition is less than 45%.
 87. The method according to claim86, wherein the HPVLP in step a) are present at an amount of 0.04 μg,and a concentration of 0.4 μg/ml.
 88. The method according to claim 86,wherein one or both of the following are met: (i) 20 to 60 ngs of HPVLPare disposed on said substrate and (ii) the ratio of sample to substrateis between 1:50 and 1:30.
 89. The method according to claim 86, whereinsaid sample is a serum sample, a urine sample, a plasma sample, a bloodsample or a cerebrospinal fluid (CSF) sample.
 90. The method accordingto claim 86, wherein said serum sample in step a) is diluted 1:101 priorto forming the first reaction mixture.
 91. The method according to claim86, wherein the second aliquot of said sample in step b) is provided ata 1:100 or 1:101 dilution.
 92. The method according to claim 86, whereinsolution-phase HPVLP in step b) is provided at a concentration of 0.4ug/ml.
 93. The method according to claim 86, wherein the labeleddetection reagent used in step b) is similarly used in step c) and stepd) to detect the level of unbound antibody in said second reactionmixture and said third reaction mixture, respectively, by detectinganti-JCV antibody capable of binding with a substrate on which isdisposed HPVLP; the labeled detection reagent comprises horseradishperoxidase (HRP); and the labeled detection reagent is diluted 1:15,000to 1:30,000.
 94. The method according to claim 93, wherein the labeleddetection reagent is diluted to about 1:20,000.
 95. The method accordingto claim 93, wherein an incubation time with the labeled detectionreagent is about 30 minutes.
 96. The method according to claim 93,wherein detecting the level of unbound antibody in said second reactionmixture in step c), and detecting the level of unbound antibody in saidthird reaction mixture in step d) further comprises: incubation with anHRP substrate for 20 minutes±2 minutes.
 97. The method according toclaim 86, wherein the sample and solution-phase HPVLP in said secondreaction mixture in step c) and said third reaction mixture in step d)are incubated for between 10-20 minutes prior to detecting the level ofunbound antibody said section reaction mixture and said third reactionmixture.
 98. The method according to claim 86, wherein the cut-offcalibrator and the positive control each comprise a mixture of serumpositive for anti-JCV antibodies and serum negative for anti-JCVantibodies; and wherein the negative control comprises serum negativefor anti-JCV antibodies.
 99. The method according to claim 86, furthercomprising acquiring the result of the assay prior to administration ofan anti-VLA-4 therapy.
 100. The method according to claim 86, furthercomprising acquiring the result of the assay after the patient has begunan anti-VLA-4 therapy.
 101. The method according to claim 86, furthercomprising determining that the patient has received treatment with ananti-VLA-4 therapy for less than 24 months.
 102. The method according toclaim 86, further comprising determining that the patient has notreceived a prior non-anti-VLA-4 immunosuppressant therapy.
 103. Themethod according to claim 102, wherein the prior non-anti-VLA-4immunosuppressant therapy is selected from beta-interferon, glatirameracetate, mitoxantrone, methotrexate, azathioprine, cyclophosphamide,mycophenolate, anti-CD20 therapy, anti-CD11a therapy, and mycophenolatemofetil.
 104. The method according to claim 86, wherein the anti-VLA-4therapy is natalizumab.
 105. The method according to claim 86, furthercomprising acquiring the result of the assay at 3 month, 6 month or 12month intervals, wherein an increase in the value indicative of thelevel of anti-JCV antibody indicates an increase in the patient's riskof developing progressive multifocal leukoencephalopathy (PML).